Optimized proteins that target Ep-CAM

ABSTRACT

Humanized Ep-CAM-targeting antibodies and methods of making and using the same are provided.

This application claims benefit under 35 U.S.C. §119(e) to U.S. Ser. No.60/697,768 filed Jul. 8, 2005, U.S. Ser. No. 60/741,966 filed Dec. 2,2005, U.S. Ser. No. 60/779,961 filed Mar. 6, 2006, and U.S. Ser. No.60/745,078 filed Apr. 18, 2006, each of which is expressly incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to optimized proteins that target theepithelial cell adhesion molecule (Ep-CAM), and their applications,particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION

Epithelial cell adhesion molecule, also known as epithelial glycoprotein40 [EGP40], epithelial protein 2 [EGP-2], GA733-2, ESA, KSA, 17-1Aantigen or other names) is an epithelial transmembrane protein encodedby the GA 733-2 gene (Gottlinger, H. G. et al. 1986, Int. J. Cancer. 15:47-53; Linnenbach, A. J. et al. 1989, Proc. Natl. Acad. Sci. USA.86:27-31; Armstrong, A. and Eck, S. 2003. Cancer Biol. Ther. 2: 320-325,Linnenbach, A. J. et al. 1993. Mol. Cel. Biol. 13:1507-1515; allexpressly incorporated by reference). The current model of the tertiaryextracellular structure of Ep-CAM indicates the presence of threedomains, including an N-terminal EGF-like domain (Armstrong, A. and Eck,S. 2003. Cancer Biol. Ther. 2: 320-325, expressly incorporated byreference), Ep-CAM is present in some normal and most neoplasticephitelial cells (Armstrong, A. and Eck, S. 2003. Cancer Biol. Ther. 2:320-325). Most carcinomas express Ep-CAM on their surfaces, includingbreast cancer, ovarian carcinoma, uterus cervix cancer, prostate cancer,kidney cancer, lung cancer, and colon cancer (Drapkin R. et al. 2004.Hum. Pathology. 35: 1014-1021; Gastl G. et al. 2000. The Lancet. 356:1981-1982; Osta, W. et al. 2004. Cancer Res. 64: 5818-5824; Went, P. T.H. et al. 2004. Hum. Pathology. 35: 122-128; all expressly incorporatedby reference). The GA733-2 gene is expressed on the baso-lateral cellsurface in most human normal epithelium (Litvinov et al. 1994. J. CellBiol. 125: 437-446, expressly incorporated by reference). It has beenpostulated that the differential localization of Ep-CAM in normal cells(baso-lateral surface) as compared with cancer cells, accounts forlimited in vivo accessibility of Ep-CAM in normal tissues (McLaughlin etal. 2001. Cancer Res. 61: 4105-4111, expressly incorporated byreference).

Monoclonal antibodies are a common class of therapeutic proteins. Anumber of favorable properties of antibodies, including but not limitedto specificity for target, ability to mediate immune effectormechanisms, and long half-life in serum, make antibodies powerfultherapeutics. A number of antibodies that target Ep-CAM have beenevaluated in pre-clinical studies with cell lines and/or xenograftmodels or in clinical trials for the treatment of cancers. Theseanti-EpCAM antibodies include but are not limited to MT201 (HD69 oradecatumumab; Naundorf, S. 2002. Int. J. Cancer. 100; 101-110; Prang, N.et al. 2005. Br. J. Cancer. 92: 342-349; Raum, T. et al. 2001. CancerImmunol. Immunother. 50: 141-150), UBS-54 (Huls et al. 1999. NatureBiotech. 17: 276-281), Edrecolomab (Panorex or Mab 17-1A; Punt et al.2002. The Lancet. 360: 671-677; Veronese, M. L. et al. 2004. Eur. J.Cancer. 40: 1229-1301; Schwartzberg, L. S. 2001. Critical Rev.Oncol./Hematol. 40: 17-24), and chimeric 17-1A mAb (LoBuglio, A. 1989.Proc. Natl. Acad. Sci. USA. 86: 4220-4224); all expressly incorporatedby reference.

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up ofindividual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins. Each chain is made up of twodistinct regions, referred to as the variable and constant regions. Thelight and heavy chain variable regions show significant sequencediversity between antibodies, and are responsible for binding the targetantigen. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. In humans there are five different classes ofantibodies including IgA (which includes subclasses IgA1 and IgA2), IgD,IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), andIgM. The distinguishing features between these antibody classes aretheir constant regions, although subtler differences may exist in the Vregion. IgG antibodies are tetrameric proteins composed of two heavychains and two light chains. The IgG heavy chain is composed of fourimmunoglobulin domains linked from N- to C-terminus in the orderV_(H)-CH1-CH2-CH3, referring to the heavy chain variable domain, heavychain constant domain 1, heavy chain constant domain 2, and heavy chainconstant domain 3 respectively (also referred to as V_(H)-Cγ1-Cγ2-Cγ3,referring to the heavy chain variable domain, constant gamma 1 domain,constant gamma 2 domain, and constant gamma 3 domain respectively). TheIgG light chain is composed of two immunoglobulin domains linked from N-to C-terminus in the order V_(L)-C_(L), referring to the light chainvariable domain and the light chain constant domain respectively.

The variable region of an antibody contains the antigen bindingdeterminants of the molecule, and thus determines the specificity of anantibody for its target antigen. The variable region is so named becauseit is the most distinct in sequence from other antibodies within thesame class. The majority of sequence variability occurs in thecomplementarity determining regions (CDRs). There are 6 CDRs total,three each per heavy and light chain, designated V_(H) CDR1, V_(H) CDR2,V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, and V_(L) CDR3. The variable regionoutside of the CDRs is referred to as the framework (FR) region.Although not as diverse as the CDRs, sequence variability does occur inthe FR region between different antibodies. Overall, this characteristicarchitecture of antibodies provides a stable scaffold (the FR region)upon which substantial antigen binding diversity (the CDRs) can beexplored by the immune system to obtain specificity for a broad array ofantigens. A number of high-resolution structures are available for avariety of variable region fragments from different organisms, someunbound and some in complex with antigen. The sequence and structuralfeatures of antibody variable regions are well characterized (Morea etal., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267-279,expressly incorporated by reference), and the conserved features ofantibodies have enabled the development of a wealth of antibodyengineering techniques (Maynard et al., 2000, Annu Rev Biomed Eng2:339-376, expressly incorporated by reference). Fragments comprisingthe variable region can exist in the absence of other regions of theantibody, including for example the antigen binding fragment (Fab)comprising V_(H)-Cγ1 and V_(H)-C_(L), the variable fragment (Fv)comprising V_(H) and V_(L), the single chain variable fragment (scFv)comprising V_(H) and V_(L) linked together in the same chain, as well asa variety of other variable region fragments (Little et al., 2000,Immunol Today 21:364-370, expressly incorporated by reference).

The Fc region of an antibody interacts with a number of Fc receptors andligands, imparting an array of important functional capabilitiesreferred to as effector functions. For IgG the Fc region comprises Igdomains Cγ2 and Cγ3 and the N-terminal hinge leading into Cγ2. Animportant family of Fc receptors for the IgG class are the Fc gammareceptors (FcγRs). These receptors mediate communication betweenantibodies and the cellular arm of the immune system (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu RevImmunol 19:275-290; both expressly incorporated by reference). In humansthis protein family includes FcγRI (CD64), including isoforms FcγRIa,FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,expressly incorporated by reference). These receptors typically have anextracellular domain that mediates binding to Fc, a membrane spanningregion, and an intracellular domain that may mediate some signalingevent within the cell. These receptors are expressed in a variety ofimmune cells including monocytes, macrophages, neutrophils, dendriticcells, eosinophils, mast cells, platelets, B cells, large granularlymphocytes, Langerhans' cells, natural killer (NK) cells, and T cells.Formation of the Fc/FcγR complex recruits these effector cells to sitesof bound antigen, typically resulting in signaling events within thecells and important subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocytosis,and cytotoxic attack. The ability to mediate cytotoxic and phagocyticeffector functions is a potential mechanism by which antibodies destroytargeted cells. The cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell is referred to as antibodydependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, AnnuRev Ceal Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290; allexpressly incorporated by reference). The cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause phagocytosis of the target cellis referred to as antibody dependent cell-mediated phagocytosis (ADCP).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65, expressly incorporated by reference). All FcγRs bind the sameregion on IgG Fc, yet with different affinities: the high affinitybinder FcγRI has a Kd for IgG1 of 10⁻⁸ M⁻¹, whereas the low affinityreceptors FcγRII and FcγRIII generally bind at 10⁻⁶ and 10⁻⁵respectively. The extracellular domains of FcγRIIIa and FcγRIIIb are 96%identical, however FcγRIIIb does not have an intracellular signalingdomain. Furthermore, whereas FcγRI, FcγRIIa/c, and FcγRIIIa are positiveregulators of immune complex-triggered activation, characterized byhaving an intracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM), FcγRIIb has an immunoreceptor tyrosine-basedinhibition motif (ITIM) and is therefore inhibitory. Thus the former arereferred to as activation receptors, and FcγRIIb is referred to as aninhibitory receptor. The receptors also differ in expression pattern andlevels on different immune cells. Yet another level of complexity is theexistence of a number of FcγR polymorphisms in the human proteome. Aparticularly relevant polymorphism with clinical significance isV158/F158 FcγRIIIa. Human IgG1 binds with greater affinity to the V158allotype than to the F158 allotype. This difference in affinity, andpresumably its effect on ADCC and/or ADCP, has been shown to be asignificant determinant of the efficacy of the anti-CD20 antibodyrituximab (Rituxan®, a registered trademark of IDEC PharmaceuticalsCorporation). Patients with the V158 allotype respond favorably torituximab treatment; however, patients with the lower affinity F158allotype respond poorly (Cartron et al., 2002, Blood 99:754-758,expressly incorporated by reference). Approximately 10-20% of humans areV158/V158 homozygous, 45% are V158/F158 heterozygous, and 35-45% ofhumans are F158/F158 homozygous (Lehrnbecher et al., 1999, Blood94:4220-4232; Cartron et al., 2002, Blood 99:754-758; both expresslyincorporated by reference). Thus 80-90% of humans are poor responders,that is they have at least one allele of the F158 FcγRIIIa.

An overlapping but separate site on Fc, serves as the interface for thecomplement protein C1q. In the same way that Fc/FcγR binding mediatesADCC, Fc/C1q binding mediates complement dependent cytotoxicity (CDC). Asite on Fc between the Cγ2 and Cγ3 domains, mediates interaction withthe neonatal receptor FcRn, the binding of which recycles endocytosedantibody from the endosome back to the bloodstream (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu RevImmunol 18:739-766; both expressly incorporated by reference). Thisprocess, coupled with preclusion of kidney filtration due to the largesize of the full length molecule, results in favorable antibody serumhalf-lives ranging from one to three weeks. Binding of Fc to FcRn alsoplays a key role in antibody transport. The binding site for FcRn on Fcis also the site at which the bacterial proteins A and G bind. The tightbinding by these proteins is typically exploited as a means to purifyantibodies by employing protein A or protein G affinity chromatographyduring protein purification. A key feature of the Fc region is theconserved N-linked glycosylation that occurs at N297. This carbohydrate,or oligosaccharide as it is sometimes referred, plays a criticalstructural and functional role for the antibody, and is one of theprinciple reasons that antibodies must be produced using mammalianexpression systems.

In addition to antibodies, an antibody-like protein that is finding anexpanding role in research and therapy is the Fc fusion (Chamow et al.,1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr OpinImmunol 9:195-200; both expressly incorporated by reference). An Fcfusion is a protein wherein one or more polypeptides is operably linkedto Fc. An Fc fusion combines the Fc region of an antibody, and thus itsfavorable effector functions and pharmacokinetics, with thetarget-binding region of a receptor, ligand, or some other protein orprotein domain. The role of the latter is to mediate target recognition,and thus it is functionally analogous to the antibody variable region.Because of the structural and functional overlap of Fc fusions withantibodies, the discussion on antibodies in the present inventionextends directly to Fc fusions.

There are a number of possible mechanisms by which antibodies destroytumor cells, including anti-proliferation via blockage of needed growthpathways, intracellular signaling leading to apoptosis, enhanced downregulation and/or turnover of receptors, CDC, ADCC, ADCP, and promotionof an adaptive immune response (Cragg et al., 1999, Curr Opin Immunol11:541-547; Glennie et al., 2000, Immunol Today 21:403-410; bothexpressly incorporated by reference). Anti-tumor efficacy may be due toa combination of these mechanisms, and their relative importance inclinical therapy appears to be cancer dependent. Despite this arsenal ofanti-tumor weapons, the potency of curretly available antibodies asanti-cancer agents is unsatisfactory, particularly given their highcost. Patient tumor response data show that monoclonal antibodiesprovide only a small improvement in therapeutic success over normalsingle-agent cytotoxic chemotherapeutics. For example, just half of allrelapsed low-grade non-Hodgkin's lymphoma patients respond to theanti-CD20 antibody rituximab (McLaughlin et al., 1998, J Clin Oncol16:2825-2833, expressly incorporated by reference). Of 166 clinicalpatients, 6% showed a complete response and 42% showed a partialresponse, with median response duration of approximately 12 months.Trastuzumab (Herceptin®, a registered trademark of Genentech), ananti-HER2/neu antibody for treatment of metastatic breast cancer, hasless efficacy. The overall response rate using trastuzumab for the 222patients tested was only 15%, with 8 complete and 26 partial responsesand a median response duration and survival of 9 to 13 months (Cobleighet al., 1999, J Clin Oncol 17:2639-2648, expressly incorporated byreference). Despite the fact that Ep-CAM is expressed on up to 77percent of colorectal cancer tumors, combination therapy with cetuximab(Erbitux®, Imclone/BMS) had an objective response rate of 22.5% with amedian duration of response of 84 days (Saltz et al., 2001, Proc. Am.Soc. Clin. Oncol. 20, 3a); results of the cetuximab single agenttreatment group were even worse. Currently for anticancer therapy, anysmall improvement in mortality rate defines success. Thus there is asignificant need to enhance the capacity of antibodies to destroytargeted cancer cells.

A promising means for enhancing the anti-tumor potency of antibodies isvia enhancement of their ability to mediate cytotoxic effector functionssuch as ADCC, ADCP, and CDC. The importance of FcγR-mediated effectorfunctions for the anti-cancer activity of antibodies has beendemonstrated in mice (Clynes et al., 1998, Proc Natl Acad Sci U S A95:652-656; Clynes et al., 2000, Nat Med 6:443-446; both expresslyincorporated by reference), and the affinity of interaction between Fcand certain FcγRs correlates with targeted cytotoxicity in cell-basedassays (Shields et al., 2001, J Biol Chem 276:6591-6604; Presta et al.,2002, Biochem Soc Trans 30:487-490; Shields et al., 2002, J Biol Chem277:26733-26740; all expressly incorporated by reference). Additionally,a correlation has been observed between clinical efficacy in humans andtheir allotype of high (V158) or low (F158) affinity polymorphic formsof FcγRIIIa (Cartron et al., 2002, Blood 99:754-758; Weng & Levy, 2003,Journal of Clinical Oncology, 21:3940-3947; both expressly incorporatedby reference). Together these data suggest that an antibody that isoptimized for binding to certain FcγRs may better mediate effectorfunctions and thereby destroy cancer cells more effectively in patients.The balance between activating and inhibiting receptors is an importantconsideration, and optimal effector function may result from an antibodythat has enhanced affinity for actvation receptors, for example FcγRI,FcγRIIa/c, and FcγRIIIa, yet reduced affinity for the inhibitoryreceptor FcγRIIb. Furthermore, because FcγRs can mediate antigen uptakeand processing by antigen presenting cells, enhanced FcγR affinity mayalso improve the capacity of antibody therapeutics to elicit an adaptiveimmune response. With respect to Ep-CAM, ADCC has been implicated as animportant effector mechanism for the anti-tumor cytotoxic capacity ofsome anti-Ep-CAM antibodies (Bleeker et al., 2004, J Immunol.173(7):4699-707; Bier et al., 1998, Cancer Immunol Immunother46:167-173, both expressly incorporated by reference).

Mutagenesis studies have been carried out on Fc towards various goals,with substitutions typically made to alanine (referred to as alaninescanning) or guided by sequence homology substitutions (Duncan et al.,1988, Nature 332:563-564; Lund et al., 1991, J Immunol 147:2657-2662;Lund et al., 1992, Mol Immunol 29:53-59; Jefferis et al., 1995, ImmunolLett 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al.,1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Shieldset al., 2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, ImmunolLett 82:57-65; U.S. Pat. Nos. 5,624,821; 5,885,573; PCT WO 00/42072; PCTWO 99/58572; all expressly incorporated by reference). Mostsubstitutions reduce or ablate binding with FcγRs. However some successhas been achieved at obtaining Fc variants with selectively enhancedbinding to FcγRs, and in some cases these Fc variants have been shown toprovide enhanced potency and efficacy in cell-based effector functionassays. See for example U.S. Pat. No. 5,624,821, PCT WO 00/42072, U.S.Pat. No. 6,737,056, U.S. Ser. No. 10/672,280, PCT US03/30249, and U.S.Ser. No. 10/822,231, and U.S. Ser. No. 60/627,774, filed Nov. 12, 2004and entitled “Optimized Fc Variants”; all expressly incorporated byreference. Enhanced affinity of Fc for FcγR has also been achieved usingengineered glycoforms generated by expression of antibodies inengineered or variant cell lines (Umaña et al., 1999, Nat Biotechnol17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shieldset al., 2002, J Bio Chem 277:26733-26740; Shinkawa et al., 2003, J BiolChem 278:3466-3473; all expressly incorporated by reference).

The present invention provides variants of Ep-CAM targeting proteinsthat comprise one or more amino acid modifications that provide enhancedeffector function and humanized light and heavy variable regions.

SUMMARY OF THE INVENTION

The present invention is directed to humanized Ep-CAM-targetingantibodies including first and/or second amino acid sequencescorresponding to the heavy and light chains of the antibodies,respectively, as well as methods of using the same. In various aspects,the first and second amino acid sequences can include sequencescorresponding to CDR3, CDR2, or CDR1 of the humanized Ep-CAM antibodyheavy and light chains. Such sequences can be independent, or can becombined.

In a first aspect, the first and second amino acid sequences comprise asequence corresponding to CDR3 of the humanized Ep-CAM heavy and lightchains. In one embodiment, the present invention is directed to ahumanized anti-Ep-CAM antibody, wherein said antibody comprises A) afirst amino acid sequence comprising i) DGPWX₁AY (SEQ ID NO:160),wherein X₁ is selected from the group consisting of F and Y; or ii) asequence selected from the group consisting of SEQ ID NOS:129-130;and/or B) a second amino acid sequence comprising i) X₁YSYPYT (SEQ IDNO:161), wherein X₁ is selected from the group consisting of G and Y; orii) a sequence selected from the group consisting of SEQ ID NOS:135-136.In certain variations, these sequences correspond to CDR3 of the heavyand light chains of the antibody.

In a further aspect, the first amino acid sequence further comprises anamino acid sequence of i) X₁X₂FX₃X₄YL (SEQ ID NO:162), wherein X₁ isselected from the group consisting of Y and F; X₂ is selected from thegroup consisting of A and S; X₃ is selected from the group consisting ofT and S; and X₄ is selected from the group consisting of N and D; andii) a sequence selected from the group consisting of SEQ ID NOS:122-126;iii) NPGSGX₁ (SEQ ID NO:163), wherein X₁ is selected from the groupconsisting of G and A; iv) the sequence of SEQ ID NOS:131-132. Thesecond amino acid sequence further comprises i) X₁NVVTY (SEQ ID NO:164),wherein X₁ is selected from the group consisting of E and Q; ii) asequence selected from the group consisting of SEQ ID NOS: 127-128; iii)X₁ASNRYT (SEQ ID NO:165), wherein X₁ is selected from the groupconsisting of G and D; or iv) an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 133-134. In certain variations, thesesequences correspond to CDR1 and CDR2 of the heavy and light chains ofthe antibody.

In a further aspect, the first and second amino acid sequences part ofthe same amino acid sequence. In a still further aspect, the first aminoacid sequence does not comprise a sequence selected from the groupconsisting of SEQ ID NOS: 122, 127, and 129, and said second amino acidsequence does not comprise a sequence selected from the group consistingof SEQ ID NOS: 131, 133, and 135.

In certain variations, the first amino acid sequence does not compriseSEQ ID NO:1, and the second amino acid sequence does not comprise SEQ IDNO:105.

In another embodiment, the humanized, the heavy chain variable regioncomprises a heavy chain framework region selected from the frameworkregions found in the group consisting of SEQ ID NOS:2-104. The secondamino acid comprises a light chain framework region selected from theframework regions found in the group consisting of SEQ ID NOS:106-121.

In another aspect, the antibody comprises a heavy chain variable regionselected from the group consisting of: SEQ ID NOS: 3, 15, 27, 56 and 97,and/or the light chain variable region of SEQ ID NO:108.

In a further aspect, the first amino acid sequence is selected from thegroup consisting of SEQ ID NOS: 2-104, and the second amino acidsequence is selected from the group consisting of SEQ ID NOS: 106-121.

In a further aspect, the antibody has an IgG1 Fc domain, or a hybridIgG1, IgG2 Fc domain.

In another aspect, the present invention is directed to a variantanti-Ep-CAM antibody comprising a variant human Fc domain, the varianthuman Fc domain comprising at least one modification that alters bindingof the antibody to an Fc receptor compared to a parent human Fc domain.In one aspect, the one modification alters binding to an Fcgammareceptor. In certain variations, the modification comprises at least onesubstitution selected from the group consisting of: 236A, 239D, 268E,298A, 298D, 326D, 326E, 330L, 330Y, 332E, 333A, 334A, and 396L, whereinthe numbering is according to the EU index in Kabat et al.

In certain variations, the modification includes an altered glycoform,such as defucosylation or lacking a fucose moiety.

In certain additional variations, the modifications can alter binding toFcRn.

In certain aspects, the variant anti-Ep-CAM antibody comprise at leastone modification that alters an effector function of the variantantibody compared to an unmodified anti-Ep-CAM antibody. In certainvariations, the effector function is antibody-dependent cellularcytotoxicity (ADCC) or complement-dependent cytoxicity (CDC).

In certain aspects, the Fc substitution comprises a substitutionselected from the group consisting of: 239D and 332E, wherein thenumbering is that of the EU index in Kabat et al. In certain otheraspects, the anti-Ep-CAM antibody comprises an Fc domain comprising atleast one substitution selected from the group consisting of: K326W,K326Y, and E333S, wherein the numbering is according to the EU index inKabat et al.

In further aspects, the modification increases the affinity of theantibody for FcγRIIIa compared to a parent antibody. In some variations,the modification increases the affinity of the antibody for FcγRIIIa atleast 2-fold compared to a parent antibody. In other variations themodification increases the affinity of the antibody for FcγRIIIa atleast 5-fold compared to a parent antibody.

In further aspects, the Fc modification decreases the affinity of theantibody for FcγRIIIa compared to a parent antibody. In someembodiments, the modification decreases the affinity of the antibody forFcγRIIIa by at least 10-fold compared to a parent antibody. Themodification can also comprise a substitution selected from the groupconsisting of: 235G and 236R, wherein the numbering is that of the EUindex in Kabat et al.

In further aspects, the Fc modification increases the FcγRIIa:FcγRIIbspecificity for the antibody. In some embodiments, the modificationincreases the FcγRIIa:FcγRIIb specificity for the antibody by at least2. In further embodiments, the modification increases theFcγRIIa:FcγRIIb specificity for the antibody by at least 8. In stillfurther embodiments, the modification increases the FcγRIIa:FcγRIIbspecificity between 7 to 11.

In other aspects, the Fc modification specifically increases maturationor activation of monocytes, macrophages, neutrophils, or dendritic cellsby the antibody compared to activation of natural killer (NK) cells bythe antibody. In some variations, the modification specificallyincreases activation of neutrophils by the antibody compared toactivation of natural killer (NK) cells by the antibody.

In other aspects, the Fc modification not substantially increaseactivation of natural killer cells or specifically increases activationby the antibody of dendritic cells.

In further aspects, the modification increases binding to an activatingFc receptor and does not increase binding to FcγRIIb. In cerain aspects,the modification specifically increases monocyte or macrophagephagocytosis.

The present invention provides variant Ep-CAM targeting proteins thatare optimized for a number of therapeutically relevant properties. Avariant Ep-CAM targeting protein comprises one or more amino acidmodifications relative to a parent Ep-CAM targeting protein, wherein theamino acid modification(s) provide one or more optimized properties.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

FIGS. 1A and 1B. Sequences of the heavy chain variable region of theoriginal 17-1A antibody and select antibodies of the present inventionwith reduced potential for immunogenicity (SEQ ID NOS:1-104 and SEQ IDNOS:122-130).

FIGS. 2A and 2B. Sequences of the light chain variable region of theoriginal 17-1A antibody and select antibodies of the present inventionwith reduced potential for immunogenicity (SEQ ID NOS:105-121 and SEQ IDNOS:13-136).

FIG. 3. Sequences of the constant regions of the original 17-1A antibodyand select antibodies of the present invention (SEQ ID NOS:137-143).

FIG. 4. Expression yields of select anti-Ep-CAM antibodies of thepresent invention.

FIG. 5. SDS gels of some anti-Ep-CAM antibodies of the presentinvention.

FIG. 6. An SDS gel of an anti-Ep-CAM antibody purified after expressionin lec13 cells. The resulting antibody has an engineered glycoform, thatis, it is defucosylated.

FIG. 7. Schematic representation of the AlphaScreen™ methods used tomeasure relative binding affinity in the present study.

FIG. 8. AlphaScreen™ data showing the relative binding affinity ofantibodies of the present invention to the antigen, Ep-CAM, and toprotein A.

FIG. 9. Summary of AlphaScreen™ data showing the relative bindingproperties of antibodies of the present invention to the antigen,Ep-CAM, and to protein A.

FIG. 10. AlphaScreen™ data showing the relative binding affinity ofantibodies of the present invention to (7A) the antigen, Ep-CAM or (7B)the Fc gamma receptor IIIa (FcgRIIIaV).

FIG. 11. Summary of AlphaScreen™ data showing the relative bindingproperties of antibodies of the present invention to the antigen,Ep-CAM.

FIG. 12. Physicochemical properties of some humanized anti-Ep-CAMantibodies and controls. Humanized variable regions were expressed withhuman IgG1. H0L0 represent the variable regions of murine 17-1A.IgG1-H0L0 is a chimeric human IgG1 with mouse variable regions.IgG2a-H0L0 contains H0L0 variable domains and mouse kappa/IgG2a constantdomains.

FIG. 13. Binding measurements of anti-Ep-CAM proteins to Ep-CAM and toFcgammaRIIIa.

FIG. 14. Binding data for anti-Ep-CAM antibodies measured by surfaceplasmon resonance (SPR).

FIG. 15. Binding data for anti-Ep-CAM antibodies measured by surfaceplasmon resonance (SPR).

FIG. 16. Binding data for anti-Ep-CAM antibodies measured by surfaceplasmon resonance (SPR).

FIG. 17. Relative expression levels of Ep-CAM and Her2 on the cell linesKATO III and SkBr3.

FIG. 18. ADCC activities of anti-Ep-CAM antibodies with the KATO IIIcell line. Variable regions were expressed with human IgG1.

FIG. 19. ADCC activities of anti-Ep-CAM antibodies with the KATO IIIcell line. Variable regions were expressed with human IgG1. (A) to (C)represent variants in direct comparison with 17-1A H3L3 and 17-1A H2L3.

FIG. 20. ADCC activities of anti-Ep-CAM antibodies with (a) the LS180cell line and (b) the LS180 and HT29 cell line.

FIG. 21. Potency and binding affinity of anti-Ep-CAM monoclonalantibodies. (A) ADCC activity with the KATO III cell line; (B) Bindingto Ep-CAM; (C) Binding to FcγRIIIaV. ADCC activity was determined withthe Europium method. Binding was determined with AlphaScreen. 17-1A H2L3I332E and 17-1A H2L3 S239D/I332E are shown.

FIG. 22. Potency and binding affinity of anti-Ep-CAM monoclonalantibodies. (A) ADCC activity with the KATO III cell line, (B) Bindingto Ep-CAM; (C) Binding to FcγRIIIaV. ADCC activity was determined withthe Europium method. Binding was determined with AlphaScreen. 17-1A H3L3I332E and 17-1A H3L3 S239D/I332E are shown.

FIG. 23. ADCC activity of anti-Ep-CAM antibodies with the SkBr3 cellline. Variable regions were expressed with human IgG1.

FIG. 24. ADCC activity of anti-Ep-CAM antibodies with the KATO III cellline. Variable regions were of variants expressed with human IgG1.

FIG. 25. FcgammaRIIIa binding to an anti-Ep-CAM protein with a typicalcarbohydrate attached to an Fc domain and to an anti-Ep-CAM protein witha defucosylated Fc domain. The glycoform variant (lower panel) hasstronger binding to the Fc receptor than the protein containing thetypical carbohydrate.

FIG. 26. Binding affinity of wild-type and variant Ep-CAM-targetingantibodies to various FcγR's, Fc gamma receptors. Data shown arecollected with H3.77 and L3 variable domains. Constant regions werebased on either human IgG1 or a hybrid of human IgG1 and IgG2 sequences.The binding affinity is plotted as −log(KD) in molar units. Largernumbers demonstrate tighter binding and a change of 1 unit on theordinate demonstrates a 10-fold change in binding affinity.

FIG. 27. Binding affinities of modified Ep-CAM-targeting antibodies toFc receptors. Surface plasmon resonance measurements were used to testthe strength of binding, which is reported as KD values in molar units.Also shown are the fold-change in binding of each antibody relative tothe WT IgG1 binding affinity and the log(1/KD) values, or −log(KD),which are also plotted in FIG. 26. The relative binding of each variantto FcγRIIa compared to FcγRIIB are shown in the last column. A value ofzero shows equal binding of the antibody to FcγRIIa and FcγRIIb, whereasa value of one shows 10fold tighter binding of the antibody to FcγRIIathan to FcγRIIb.

FIG. 28. (a) Common allotypes of human IgGs. (b) Alternative allotypicversions of anti-Ep-CAM IgG antibodies.

FIG. 29. Sequences of Ep-CAM-targeting antibodies, including both heavyand light chain sequences (SEQ ID NOS:144-159).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to humanized Ep-CAM-targetingantibodies including first and/or second amino acid sequencescorresponding to the heavy and light chains of the antibodies,respectively, as well as methods of using the same. In various aspects,the first and second amino acid sequences can include sequencescorresponding to CDR3, CDR2, or CDR1 of the humanized Ep-CAM antibodyheavy and light chains. Such sequences can be independent, or can becombined. Additionally, the Ep-CAM targeting antibodies can be combinedwith variant Fc regions designed to alter effector function, includingthose of U.S. patent application Ser. No. 11/124,620 filed May 5, 2005,ser. 10/822,231 filed Mar. 26, 2004, and ser. No. 10/379,392, filed Mar.3, 2003, each of which is incorporated herein by reference in itsentirety.

In order that the invention may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phaqocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. The preferredamino acid modification herein is a substitution. By “amino acidsubstitution” or “substitution” herein is meant the replacement of anamino acid at a particular position in a parent polypeptide sequencewith another amino acid. For example, the substitution 1332E refers to avariant polypeptide, in this case an Fc variant, in which the isoleucineat position 332 is replaced with a glutamic acid.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position. By “protein” herein ismeant at least two covalently attached amino acids, which includesproteins, polypeptides, oligopeptides and peptides. The protein may bemade up of naturally occurring amino acids and peptide bonds, orsynthetic peptidomimetic structures, i.e. “analogs”, such as peptoids(see Simon et al., 1992, Proc Natl Acad Sci USA 89(20):9367)particularly when LC peptides are to be administered to a patient. Thus“amino acid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example homophenylaianine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chain may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradation.

By “affinity” or “binding affinity” as used herein is meant the strengthof interaction between two molecules. The strength of affinity is oftenreported with a dissociation constant, Kd or KD, such as 1*10⁻⁷ M, or alog(Kd), such as −7.0, or −log(Kd), such as 7.0. As is known in the art,lower values of Kd correspond to tighter binding and higher affinity.Higher values of Kd correspond to weaker binding and lower affinity.

The binding “specificity” may be defined as the relative strength ofbinding of a first molecule to a second molecule compared to thestrength of the first molecule to a third molecule. Specificity may bereported as a ratio or quotient of binding constants for the two bindingreactions. For example, a FcγRIIa:FcγRIIb specificity of 10 for AntibodyA, means that Antibody A binds to FcγRIIa ten-fold more strongly than itbinds to FcγRIIb. An additional way to express the same FcγRIIa:FcγRIIbspecificity for Antibody A is that the Kd of FcγRIIb is 10-fold higherthan the Kd of FcγRIIa.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC. By “effector cell” as used herein is meant a cellof the immune system that expresses one or more Fc receptors andmediates one or more effector functions. Effector cells include but arenot limited to monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γγ T cells, and may befrom any organism including but not limited to humans, mice, rats,rabbits, and monkeys. By “library” herein is meant a set of Fc variantsin any form, including but not limited to a list of nucleic acid oramino acid sequences, a list of nucleic acid or amino acid substitutionsat variable positions, a physical library comprising nucleic acids thatencode the library sequences, or a physical library comprising the Fcvariant proteins, either in purified or unpurified form.

By “Ep-CAM targeting protein” as used herein is meant a protein thatbinds to Ep-CAM, also known as epithelial glycoprotein 40 [EGP40],epithelial protein 2 [EGP-2], GA733-2, ESA, KSA, 17-1A antigen and othernames. The EQCAM targeting protein of the present invention may be anantibody, Fc fusion, or any other protein that binds Ep-CAM. An Ep-CAMtargeting protein of the present invention may bind any epitope orregion on Ep-CAM, and may be specific for fragments, splice forms, oraberrent forms of Ep-CAM. Preferred proteins are antibodies, includingthe antibodies described herein.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For lgG, Fc comprises immunoglobulin domainsCgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1)and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary,the human IgG heavy chain Fc region is usually defined to compriseresidues C226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. Fc may refer to this region inisolation, or this region in the context of an Fc polypeptide, asdescribed below. By “Fc polypeptide” as used herein is meant apolypeptide that comprises all or part of an Fc region. Fc polypeptidesinclude antibodies, Fc fusions, isolated Fcs, and Fc fragments.

By “Fc fusion” as used herein is meant a protein wherein one or morepolypeptides or small molecules is operably linked to an Fc region or aderivative thereof. Fc fusion is herein meant to be synonymous with theterms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptorglobulin” (sometimes with dashes) as used in the prior art (Chamow etal., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr OpinImmunol 9:195-200; both expressly incorporated by reference). An Fcfusion combines the Fc region of an immunoglobulin with a fusionpartner, which in general can be any protein or small molecule. The roleof the non-Fc part of an Fc fusion, i.e. the fusion partner, is oftenbut not always to mediate target binding, and thus it is functionallyanalogous to the variable regions of an antibody. A variety of linkers,defined and described below, may be used to covalently link Fc to afusion partner to generate an Fc fusion.

By “Fc gamma receptor” or “ScγR” as used herein is meant any member ofthe family of proteins that bind the IgG antibody Fc region and aresubstantially encoded by the FcγR genes. In humans this family includesbut is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb,and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (includingallotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2),and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,expressly incorporated by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limied to FcγRI (CD64), FcγRII (CD32),FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs or FcγR isoforms or allotypes.

By “Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of anantibody to form an Fc-ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A, streptococcal protein G, andviral FcγR. Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis etal., 2002, Immunological Reviews 190:123-136, expressly incorporated byreference). Fc ligands may include undiscovered molecules that bind Fc.

By “IgG” as used herein is meant a polypeptide belonging to the class ofantibodies that are substantially encoded by a recognized immunoglobulingamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4.In mice this class comprises IgG1, IgG2a, IgG2b, IgG3. Also included arehybrids of IgG proteins in which amino acids for one IgG proteinsubstituted for amino acids of a different IgG protein (e.g. IgG1/IgG2hybrids. By “immunoglobulin (Ig)” herein is meant a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full length antibodies, antibody fragments, and individualimmunoglobulin domains. By “immunoglobulin (Ig) domain” herein is meanta region of an immunoglobulin that exists as a distinct structuralentity as ascertained by one skilled in the art of protein structure. Igdomains typically have a characteristic β-sandwich folding topology. Theknown Ig domains in the IgG class of antibodies are V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

By “parent polypeptide” or “precursor polypeptide” (including Fc parentor precursors) as used herein is meant a polypeptide that issubsequently modified to generate a variant. The parent polypeptide maybe a naturally occurring polypeptide, or a variant or engineered versionof a naturally occurring polypeptide. Parent polypeptide may refer tothe polypeptide itself, compositions that comprise the parentpolypeptide, or the amino acid sequence that encodes it. Accordingly, by“parent Fc polypeptide” as used herein is meant a Fc polypeptide that ismodified to generate a variant, and by “parent antibody” as used hereinis meant an antibody that is modified to generate a variant antibody.

As outlined above, certain positions of the Fc molecule can be altered.By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index as in Kabat. For example,position 297 is a position in the human antibody IgG1. Correspondingpositions are determined as outlined above, generally through alignmentwith other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound.

By “target cell” as used herein is meant a cell that expresses a targetantigen,

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V_(K), Vλ, and/or V_(H) genes that make up thekappa, lambda, and heavy chain immunoglobulin genetic loci respectively.

By “variant protein”, “protein variant”, “variant polypeptide”, or“polypeptide variant” as used herein is meant a polypeptide sequencethat differs from that of a parent polypeptide sequence by virtue of atleast one amino acid modification. Variant polypeptide may refer to thepolypeptide itself, a composition comprising the polypeptide, or theamino sequence that encodes it. Preferably, the variant polypeptide hasat least one amino acid modification compared to the parent polypeptide,e.g. from about one to about ten amino acid modifications, andpreferably from about one to about five amino acid modificationscompared to the parent. The variant polypeptide sequence herein willpreferably possess at least about 80% homology with a parent polypeptidesequence, and most preferably at least about 90% homology, morepreferably at least about 95% homology. Accordingly, by “variant Fc” or“Fc variant” as used herein is meant an Fc sequence that differs fromthat of a parent Fc sequence by virtue of at least one amino acidmodification. An Fc variant may only encompass an Fc region, or mayexist in the context of an antibody, Fc fusion, or other polypeptidethat is substantially encoded by Fc. Fc variant may refer to the Fcpolypeptide itself compositions comprising the Fc variant polypeptide,or the amino acid sequence that encodes it. Also included are Fcvariants disclosed in U.S. patent application Ser. No. 11/124,620 filedMay 5, 2005, Ser. No. 10/822,231 filed Mar. 26, 2004, and Ser. No.10/379,392, filed Mar. 3, 2003, each of which is incorporated herein byreference in its entirety. Accordingly, by “variant EP-CAM targetingprotein” or “Eq-CAM targeting protein variant” as used herein is meantan Ep-CAM targeting protein, as defined above, that differs in sequencefrom that of a parent Ep-CAM targeting protein sequence by virtue of atleast one amino acid modification. Variant Ep-CAM targeting protein mayrefer to the protein itself, compositions comprising the protein, or theamino acid sequence that encodes it.

For all immunoglobulin heavy chain constant region positions discussedin the present invention, numbering is according to the EU index as inKabat (Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, 5th Ed., United States Public Health Svice, NationalInstitutes of Health, Bethesda). The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

Antibodies

Accordingly, the present invention provides variant antibodies.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. Heavy chains are classifiedas mu, delta, gamma, alpha, or epsilon, and define the antibody'sisotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has severalsubclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.IgM has subclasses, including, but not limited to, IgM1 and IgM2. Thus,“isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human inmunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. In the variable region, three loops are gathered for eachof the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al.).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobutin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions . Accordingly, “CH” domains in the context of IgG areas follows: “CH1” refers to positions 118-220 according to the EU indexas in Kabat. “CH2” refers to positions 237-340 according to the EU indexas in Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230.

Of particular interest in the present invention are the Fc regions. By“Fc” or “Fc region”, as used herein is meant the polypeptide comprisingthe constant region of an antibody excluding the first constant regionimmunoglobulin domain and in some cases, part of the hinge. Thus Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgG, and the last three constant region immunoglobulin domainsof IgE and IgM, and the flexible hinge N-terminal to these domains. ForIgA and IgM, Fc may include the J chain. For IgG, as illustrated in FIG.1, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cg2 and Cg3)and the lower hinge region between Cgamma1 (Cg1) and Cgamma2 (Cg2).Although the boundaries of the Fc region may vary, the human IgG heavychain Fc region is usually defined to include residues C226 or P230 toits carboxyl-terminus, wherein the numbering is according to the EUindex as in Kabat. Fc may refer to this region in isolation, or thisregion in the context of an Fc polypeptide, as described below. By “Fcpolypeptide” as used herein is meant a polypeptide that comprises all orpart of an Fc region. Fc polypeptides include antibodies, Fc fusions,isolated Fcs, and Fc fragments.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

Antibody Fragments

In one embodiment, the antibody is an antibody fragment. Of particularinterest are antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546) which consists of a singlevariable, (v) isolated CDR regions, (vi) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii)bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”or “triabodies”, multivalent or multispecific fragments constructed bygene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479;WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:6444-6448, each of which is incorporated herein by reference in itsentirey). The antibody fragments may be modified. For example, themolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245).

Chimeric and Humanized Antibodies

In some embodiments, the scaffold components can be a mixture fromdifferent species. For example, if the antibody is a mixture of a humanantibody and a mouse antibody, such an antibody may be a chimericantibody and/or a humanized antibody. In general, both “chimericantibodies” and “humanized antibodies” refer to antibodies that combineregions from more than one species. For example, “chimeric antibodies”traditionally comprise variable region(s) from a mouse (or rat, in somecases) and the constant region(s) from a human. “Humanized antibodies”generally refer to non-human antibodies that have had thevariable-domain framework regions swapped for sequences found in humanantibodies. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213). The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin, and thus will typically comprise a human Fc region.Humanized antibodies can also be generated using mice with a geneticallyengineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein). Humanization methods include but are not limited tomethods described in Jones et al., 1986, Nature 321:522-525; Riechmannet al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9, Presta et al., 1997, CancerRes.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:4181-4185; O'Connor at al., 1998, Protein Eng 11:321-8. Humanizationor other methods of reducing the immunogenicity of nonhuman antibodyvariable regions may include resurfacing methods, as described forexample in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973.In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al. 1997, J. Biol. Chem. 272(16):10678-10684; Rosoket al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998,Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, ProteinEngineering 16(10):753-759. Other humanization methods may involvechanging both CDR and non-CDR regions, including but not limited tomethods described in U.S. Ser. No. 09/810,502; Tan et al., 2002, J.Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.169:3076-3084.

Bispecific Antibodies

In one embodiment, the antibodies of the invention multispecificantibody, and notably a bispecitic antibody, also sometimes referred toas “diabodies”. These are antibodies that bind to two (or more)different antigens. Diabodies can be manufactured in a variety of waysknown in the art (Holliger and Winter, 1993, Current Opinion Biotechnol.4:446-449), e.g., prepared chemically or from hybrid hybridomas.

Minibodies

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scfv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061. In some cases, the scFv can bejoined to the Fc region, and may include some or all of the hingeregion.

Human Antibodies

In one embodiment, the antibody is a fully human antibody with at leastone modification as outlined herein. “Fully human antibody ” or“complete human antibody” refers to a human antibody having the genesequence of an antibody derived from a human chromosome with themodifications outlined herein.

Antibody Fusions

In one embodiment, the antibodies of the invention are antibody fusionproteins (sometimes referred to herein as an “antibody conjugate”). Onetype of antibody fusions are Fc fusions, which join the Fc region with aconjugate partner. By “Fc fusion” as used herein is meant a proteinwherein one or more polypeptides is operably linked to an Fc region. Fcfusion is herein meant to be synonymous with the terms “immunoadhesin”,“Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes withdashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fcfusion combines the Fc region of an immunoglobulin with a fusionpartner, which in general can be any protein or small molecule.Virtually any protein or small molecule may be linked to Fc to generatean Fc fusion. Protein fusion partners may include, but are not limitedto, the variable region of any antibody, the target-binding region of areceptor, an adhesion molecule, a ligand, an enzyme, a cytokine, achemokine, or some other protein or protein domain. Small moleculefusion partners may include any therapeutic agent that directs the Fcfusion to a therapeutic target. Such targets may be any molecule,preferably an extracellular receptor, that is implicated in disease.

In addition to Fc fusions, antibody fusions include the fusion of theconstant region of the heavy chain with one or more fusion partners(again including the variable region of any antibody), while otherantibody fusions are substantially or completely full length antibodieswith fusion partners. In one embodiment, a role of the fusion partner isto mediate target binding, and thus it is functionally analogous to thevariable regions of an antibody (and in fact can be). Virtually anyprotein or small molecule may be linked to Fc to generate an Fc fusion(or antibody fusion). Protein fusion partners may include, but are notlimited to, the target-binding region of a receptor, an adhesionmolecule, a ligand, an enzyme, a cytokine, a chemokine, or some otherprotein or protein domain. Small molecule fusion partners may includeany therapeutic agent that directs the Fc fusion to a therapeutictarget. Such targets may be any molecule, preferably an extracellularreceptor, that is implicated in disease.

The conjugate partner can be proteinaceous or non-proteinaceous; thelatter generally being generated using functional groups on the antibodyand on the conjugate partner. For example linkers are known in the art;for example, homo-or hetero-bifunctional linkers as are well known (see,1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs; for the latter, see U.S.2003/0050331, hereby incorporated by reference in its entirety.

Covalent Modifications of Antibodies

Covalent modifications of (e.g. attachments to) antibodies are includedwithin the scope of this invention, and are generally, but not always,done post-translationally. For example, several types of covalentattachments to the antibody are introduced into the molecule by reactingspecific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-aminocontaining residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid:O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or 131I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-mateimido1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryi or threonyl residues,methytation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Glycosylation

Another type of covalent modification is glycosylation. In anotherembodiment, the IgG variants disclosed herein can be modified to includeone or more engineered glycoforms. By “engineered glycoform” as usedherein is meant a carbohydrate composition that is covalently attachedto an IgG, wherein said carbohydrate composition differs chemically fromthat of a parent IgG. Engineered glycoforms may be useful for a varietyof purposes, including but not limited to enhancing or reducing effectorfunction. Engineered glycoforms may be generated by a variety of methodsknown in the art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davieset al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473);(U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCTWO 02/30954A1); (Potelligent™ technology [Biowa, Inc., Princeton, N.J.];GlycoMAb™ glycosylation engineering technology [GLYCART biotechnologyAG, Zürich, Switzerland]). Many of these techniques are based oncontrolling the level of fucosylated and/or bisecting oligosaccharidesthat are covalently attached to the Fc region, for example by expressingan IgG in various organisms or cell lines, engineered or otherwise (forexample Lec-13 CHO cells or rat hybridoma YB2/0 cells), by regulatingenzymes involved in the glycosylation pathway (for example FUT8[α1,6-fucosyltranserase] and/or β1-4-N-acetylglucosaminyltransferase III[GnTIII]), or by modifying carbohydrate(s) after the IgG has beenexpressed. Engineered glycoform typically refers to the differentcarbohydrate or oligosaccharide; thus an IgG variant, for example anantibody or Fc fusion, can include an engineered glycoform.Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are frequently the recognition sequences forenzymatic attachment of the carbohydrate moiety to the asparagine sidechain. Thus. the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be preventedby the use of the compound tunicamycin as described by Duskin et al.,1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation ofprotein-N-glycoside linkages. Additionally, modification of an aminoacid in the glycosylation motif may be used to prevent glycosylation.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037, incorporated herein by reference in its entirety.

Labeled Antibodies

In some embodiments, the covalent modification of the antibodies of theinvention comprises the addition of one or more labels. In some cases,these are considered antibody fusions.

The term “labelling group” means any detectable label. In someembodiments, the labelling group is coupled to the antibody via spacerarms of various lengths to reduce potential steric hindrance. Variousmethods for labelling proteins are known in the art and may be used inperforming the present invention.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and may be used in performing the present invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995,5,925,558). All of the above-cited references are expressly incorporatedherein by reference.

In certain variations, antibody may mean a protein consisting of one ormore polypeptides substantially encoded by all or part of the recognizedimmunoglobulin genes. The recognized immunoglobulin genes, for examplein humans, include the kappa (κ), lambda (λ), and heavy chain geneticloci, which together comprise the myriad variable region genes, and theconstant region genes mu (υ), delta (δ), gamma (γ), sigma (ε), and alpha(α) which encode the IgM, IgD, IgG (IgG1, IgG2, IgG3, and IgG4), IgE,and IgA (IgA1 and IgA2) isotypes respectively. Antibody herein is meantto include full length antibodies and antibody fragments, and may referto a natural antibody from any organism, an engineered antibody, or anantibody generated recombinantly for experimental, therapeutic, or otherpurposes.

EF-CAM Targeting Proteins

The Ep-CAM targeting proteins of the present invention may be anantibody, referred to herein as “anti-Ep-CAM antibodies”. Anti-Ep-CAMantibodies of the present invention may comprise immunoglobulinsequences that are substantially encoded by immunoglobulin genesbelonging to any of the antibody classes, including but not limited toIgG (including human subclasses IgG1, IgG2, IgG3, or IgG4 and hybridsthereof), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG,and IgM classes of antibodies. Most preferably the antibodies of thepresent invention comprise sequences belonging to the human IgG class ofantibodies. Anti-Ep-CAM antibodies of the present invention may benonhuman, chimeric, humanized, or fully human. As will be appreciated byone skilled in the art, these different types of antibodies reflect thedegree of “humaness” or potential level of immunogenicity in a human.For a description of these concepts, see Clark et al., 2000 andreferences cited therein (Clark, 2000, Immunol Today 21:397-402,expressly incorporated by reference). Chimeric antibodies comprise thevariable region of a nonhuman antibody, for example VH and VL domains ofmouse or rat origin, operably linked to the constant region of a humanantibody (see for example U.S. Pat. No. 4,816,567, expresslyincorporated by reference). The nonhuman variable region may be derivedfrom any organism as described above, preferably mammals and mostpreferably rodents or primates. In one embodiment, the antibody of thepresent invention comprises monkey variable domains, for example asdescribed in Newman et al., 1992, Biotechnology 10:1455-1460, U.S. Pat.Nos. 5,658,570, and 5,750,105; all expressly incorporated by reference.In a preferred embodiment, the variable region is derived from anonhuman source, but its immunogenicity has been reduced using proteinengineering. In a preferred embodiment, the antibodies of the presentinvention are humanized (Tsurushita & Vasquez, 2004, Humanization ofMonoclonal Antibodies, Molecular Biology of B Cells, 533-545, ElsevierScience (USA), expressly incorporated by reference). By “humanized”antibody as used herein is meant an antibody comprising frameworkregions (FRs) derived from one or more human sequences and one or morecomplementarity determining regions (CDR's) from a non-human (usuallymouse or rat) antibody. One common method used in the art is called “CDRgrafting” in which a non-human antibody (the “donor”) provides the CDR'sand a human antibody (the “acceptor”) provides the frameworks (WinterU.S. Pat. No. 5,225,539). In CDR grafting “backmutation” of selectedacceptor framework residues to the corresponding donor residues is oftenrequired to regain affinity that is lost in the initial graftedconstruct (U.S. Pat. Nos. 5,530,101; 5,585,089, 5,693,761; 5,693,762;6,180,370, 5,859,205; 5,821,337; 6,054,297; and 6,407,213; all expresslyincorporated by reference). Optimally the humanized antibody also willcomprise at least a portion of an immunoglobulin constant region,typically that of a human immunoglobulin, and thus will typicallycomprise a human Fc region. Alternatively, in a most preferredembodiment, and as described more fully in Example 1, the immunogenicityof the antibody may be reduced using a method described in U.S. Ser. No.60/619,483, filed Oct. 14, 2004 and U.S. Ser. No. 11/004,590, entitled“Methods of Generating Variant Proteins with Increased Host StringContent and Compositions Thereof”, filed on Dec. 6, 2004; both expresslyincorporated by reference. In an alternate embodiment, the antibodies ofthe present invention may be fully human, that is the sequences of theantibodies are completely or substantially human. A number of methodsare known in the art for generating fully human antibodies, includingthe use of transgenic mice (Bruggemann et al., 1997, Curr OpinBiotechnol 8:455-458, expressly incorporated by reference) or humanantibody libraries coupled with selection methods (Griffiths et al.,1998, Curr Opin Biotechnol 9:102-108, expressly incorporated byreference).

Of particular interest are techniques that allow optimization of humanand non-human components of the antibodies, as described in U.S. Ser.No. 11/149,943 filed Jun. 9, 2005, which are expressly incorporated byreference in their entirety. Specifically, techniques are used that relyon the incorporation of different human non-CDR regions to form anon-CDR region that is composed of sequences from different humansequences (e.g. different human germline sequences); thus, these regionscomprise sequences that are “human” to the extent that all thecomponents come from human sequences. These sequences can beadditionally optimized with Fc variants, CDR variants, etc.

The Ep-CAM targeting proteins of the present invention may be an Fcfusion, referred to herein as “anti-Ep-CAM Fc fusions”. Anti-Ep-CAM Fcfusions of the present invention comprise an Fc polypeptide operablylinked to one or more fusion partners. The role of the fusion partnertypically, but not always, is to mediate binding of the Fc fusion to atarget antigen. (Chamow et al., 1996, Trends Biotechnol 14:52-60;Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; both expresslyincorporated by reference). For the present invention, one of the fusionpartners must bind Ep-CAM. Fusion partners may be a protein,polypeptide, or small molecule. Virtually any polypeptide or moleculethat targets Ep-CAM may serve as a fusion partner. Undiscovered Ep-CAMligands may serve as fusion partners for the Ep-CAM targeting proteinsof the present invention. Anti-Ep-CAM Fc fusions of the invention maycomprise immunoglobulin sequences that are substantially encoded byimmunoglobulin genes belonging to any of the antibody classes, includingbut not limited to IgG (including human subclasses IgG1, IgG2, IgG3, orIgG4), IgA (including human subclasses IgA1 and IgA2), IgD, IgE, IgG,and IgM classes of antibodies. Most preferably the anti-Ep-CAM Fcfusions of the present invention comprise sequences belonging to thehuman IgG class of antibodies.

Ep-CAM targeting proteins of the present invention, including antibodiesand Fc fusions, may comprise Fc fragments. An Fc fragment of the presentinvention may comprise from 1-90% of the Fc region, with 10-90% beingpreferred, and 30-90% being most preferred. Thus for example, an Fcfragment of the present invention may comprise an IgG1 Cγ2 domain, anIgG1 Cγ2 domain and hinge region, an IgG1 Cγ3 domain, and so forth. Inone embodiment, an Fc fragment of the present invention additionallycomprises a fusion partner, effectively making it an Fc fragment fusion.Fc fragments may or may not contain extra polypeptide sequence.

Ep-CAM targeting proteins of the present invention may be substantiallyencoded by genes from any organism, preferably mammals, including butnot limited to humans, rodents including but not limited to mice andrats, lagomorpha including but not limited to rabbits and hares caeiidaeincluding but not limited to camels, llamas, and dromedaries, andnon-human primates, including but not limited to Prosimians, Platyrrhini(New World monkeys), Cercopithecoidea (Old World monkeys), andHominoidea including the Gibbons and Lesser and Great Apes. In a mostpreferred embodiment, the Ep-CAM targeting proteins of the presentinvention are substantially human. The Ep-CAM targeting proteins of thepresent invention may be substantially encoded by immunoglobulin genesbelonging to any of the antibody classes. In a most preferredembodiment, the Ep-CAM targeting proteins of the present inventioncomprise sequences belonging to the IgG class of antibodies, includinghuman subclasses IgG1, IgG2, IgG3, and IgG4. In an alternate embodiment,the Ep-CAM targeting proteins of the present invention comprisesequences belonging to the IgA (including human subclasses IgA1 andIgA2), IgD, IgE, IgG, or IgM classes of antibodies. The Ep-CAM targetingproteins of the present invention may comprise more than one proteinchain. That is, the present invention may find use in an Ep-CAMtargeting protein that is a monomer or an oligomer, including a homo- orhetero-oligomer.

In the most preferred embodiment, the anti-Ep-CAM antibodies and Fcfusions of the invention are based on human IgG sequences, and thushuman IgG sequences are used as the “base” sequences against which othersequences are compared, including but not limited to sequences fromother organisms, for example rodent and primate sequences, as well assequences from other immunoglobulin classes such as IgA, IgE, IgGD,IgGM, and the like. It is contemplated that, although the Ep-CAMtargeting proteins of the present invention are engineered in thecontext of one parent Ep-CAM targeting protein, the variants may beengineered in or “transferred” to the context of another, second parentEp-CAM targeting protein. This is done by determining the “equivalent”or “corresponding” residues and substitutions between the first andsecond Ep-CAM targeting proteins, typically based on sequence orstructural homology between the sequences of the two Ep-CAM targetingproteins. In order to establish homology, the amino acid sequence of afirst Ep-CAM targeting protein outlined herein is directly compared tothe sequence of a second Ep-CAM targeting protein. After aligning thesequences, using one or more of the homology alignment programs known inthe art (for example using conserved residues as between species),allowing for necessary insertions and deletions in order to maintainalignment (i.e., avoiding the elimination of conserved residues througharbitrary deletion and insertion), the residues equivalent to particularamino acids in the primary sequence of the first Ep-CAM targetingprotein are defined. Alignment of conserved residues preferably shouldconserve 100% of such residues. However, alignment of greater than 75%or as little as 50% of conserved residues is also adequate to defineequivalent residues. Equivalent residues may also be defined bydetermining structural homology between a first and second Ep-CAMtargeting protein that is at the level of tertiary structure for EP-CAMtargeting proteins whose structures have been determined. In this case,equivalent residues are defined as those for which the atomiccoordinates of two or more of the main chain atoms of a particular aminoacid residue of the parent or precursor (N on N, CA on CA, C on C and Oon O) are within 0.13 nm and preferably 0.1 nm after alignment.Alignment is achieved after the best model has been oriented andpositioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the proteins. Regardless of how equivalentor corresponding residues are determined, and regardless of the identityof the parent Ep-CAM targeting protein in which the Ep-CAM targetingproteins are made, what is meant to be conveyed is that the Ep-CAMtargeting proteins discovered by the present invention may be engineeredinto any second parent Ep-CAM targeting protein that has significantsequence or structural homology with the Ep-CAM targeting protein. Thusfor example, if a variant anti-Ep-CAM antibody is generated wherein theparent anti-Ep-CAM antibody is human IgG1, by using the methodsdescribed above or other methods for determining equivalent residues,the variant anti-Ep-CAM antibody may be engineered in a human IgG2parent anti-Ep-CAM antibody, a human IgA parent anti-Ep-CAM antibody, amouse IgG2a or IgG2b parent anti-Ep-CAM antibody, and the like. Again,as described above, the context of the parent Ep-CAM targeting proteindoes not affect the ability to transfer the Ep-CAM targeting proteins ofthe present invention to other parent Ep-CAM targeting proteins. Forexample, the variant anti-Ep-CAM antibodies that are engineered in ahuman IgG1 antibody that targets one Ep-CAM epitope may be transferredinto a human IgG2 antibody that targets a different Ep-CAM epitope, intoan Fc fusion that comprises a human IgG1 Fc region that targets yet adifferent Ep-CAM epitope, and so forth.

The Ep-CAM targeting protein of the present invention may be virtuallyany antibody, Fc fusion, or other protein that binds Ep-CAM. Ep-CAMtargeting proteins of the invention may display selectivity for Ep-CAMversus alternative targets, for example other RTKs, or selectivity for aspecific form of the Ep-CAM target versus alternative forms. Examplesinclude full-length versus splice variants, cell-surface vs. solubleforms, selectivity for various polymorphic variants, or selectivity forspecific conformational forms of a target. An Ep-CAM targeting proteinof the present invention may bind any epitope or region on Ep-CAM, andmay be specific for fragments, mutant forms, splice forms, or aberrantforms of Ep-CAM.

The Ep-CAM targeting proteins of the present invention may find use in awide range of products. In one embodiment the Ep-CAM targeting proteinof the invention is a therapeutic, a diagnostic, or a research reagent.Alternatively, the Ep-CAM targeting protein of the present invention maybe used for agricultural or industrial uses, An anti-Ep-CAM antibody ofthe present invention may find use in an antibody composition that ismonoclonal or polyclonal. The Ep-CAM targeting proteins of the presentinvention may include agonists, antagonists, neutralizing, inhibitory,or stimulatory. In a preferred embodiment, the Ep-CAM targeting proteinsof the present invention are used to kill target cells that bear theEp-CAM target antigen, for example cancer cells. In an alternateembodiment, the EP-CAM targeting proteins of the present invention areused to block, antagonize, or agonize the Ep-CAM target antigen. In analternately preferred embodiment, the Ep-CAM targeting proteins of thepresent invention are used to block, antagonize, or agonize the targetantigen and kill the target cells that bear the target antigen.

Modifications

The present invention provides variant Ep-CAM targeting proteins thatare optimized for a number of therapeutically relevant properties. Avariant Ep-CAM targeting protein comprises one or more amino acidmodifications relative to a parent Ep-CAM targeting protein, wherein theamino acid modification(s) provide one or more optimized properties.Thus the Ep-CAM targeting proteins of the present invention are variantsEp-CAM targeting proteins. An Ep-CAM targeting protein of the presentinvention differs in amino acid sequence from its parent Ep-CAMtargeting protein by virtue of at least one amino acid modification.Thus variant Ep-CAM targeting proteins of the present invention have atleast one amino acid modification compared to the parent. Alternatively,the variant Ep-CAM targeting proteins of the present invention may havemore than one amino acid modification as compared to the parent, forexample from about one to fifty amino acid modifications, preferablyfrom about one to ten amino acid modifications, and most preferably fromabout one to about five amino acid modifications compared to the parent.Thus the sequences of the variant Ep-CAM targeting proteins and those ofthe parent Ep-CAM targeting proteins are substantially homologous. Forexample, the variant Ep-CAM targeting protein sequences herein willpossess about 80% homology with the parent Ep-CAM targeting proteinsequence, preferably at least about 90% homology, and most preferably atleast about 95% homology.

In a most preferred embodiment, the Ep-CAM targeting proteins of thepresent invention comprise amino acid modifications that provideoptimized effector function properties relative to the parent. Mostpreferred substitutions and optimized effector function properties aredescribed in U.S. Ser. No. 10/672,280, PCT US03/30249, and U.S. Ser. No.10/822,231, and U.S. Ser. No. 60/627,774, filed Nov. 12, 2004 andentitled “Optimized Fc Variants”. Properties that may be optimizedinclude but are not limited to enhanced or reduced affinity for an FcγR.In a preferred embodiment, the Ep-CAM targeting proteins of the presentinvention are optimized to possess enhanced affinity for a humanactivating FcγR, preferably FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, andFcγRIIIb, most preferably FcγRIIIa. In an alternately preferredembodiment, the Ep-CAM targeting proteins are optimized to possessreduced affinity for the human inhibitory receptor FcγRIIb. Thesepreferred embodiments are anticipated to provide Ep-CAM targetingproteins with enhanced therapeutic properties in humans, for exampleenhanced effector function and greater anti-cancer potency. In analternate embodiment, the Ep-CAM targeting proteins of the presentinvention are optimized to have reduced or ablated affinity for a humanFcγR, including but not limited to FcγRI, FcγRIIIa, FcγRIIb, FcγRIIc,FcγRIIIa, and FcγRIIIb. These embodiments are anticipated to provideEp-CAM targeting proteins with enhanced therapeutic properties inhumans, for example reduced effector function and reduced toxicity. Inother embodiments, Ep-CAM targeting proteins of the present inventionprovide enhanced affinity for one or more FcγRs, yet reduced affinityfor one or more other FcγRs. For example, an Ep-CAM targeting protein ofthe present invention may have enhanced binding to FcγRIIIa, yet reducedbinding to FcγRIIb. Alternately, an Ep-CAM targeting protein of thepresent invention may have enhanced binding to FcγRIIa and FcγRI, yetreduced binding to FcγRIIb. In yet another embodiment, an Ep-CAMtargeting protein of the present invention may have enhanced affinityfor FcγRIIb, yet reduced affinity to one or more activating FcγRs.

In certain embodiments, the Ep-CAM targeting proteins are anti-EpCAMantibodies that comprise an Fc variant of a human Fc polypeptide asdefined in U.S. patent application Ser. No. 11/124,620, or variationsthereof as derived in U.S. patent application Ser. No. 11/149,943. TheFc variants exhibit altered binding to an Fc ligand as compared to humanFc polypeptide. In one embodiment, the Fc variant has the formulacomprising:

-   -   Vb(221)-Vb(222)-Vb(223)-Vb(224)-Vb(225)-Fx(226)-Vb(227)-Vb(228)-Fx(229)-Vb        (230)-Vb(231)-Vb(232)-Vb(233)-Vb(234)-Vb(235)-Vb(236)-Vb(237)-Vb(238)-Vb        (239)-Vb(240)-Vb(241)-Fx(242)-Vb(243)-Vb(244)-Vb(245)-Vb(246)-Vb(247)-Fx        (248)-Vb(249)-Fx(250-254)-Vb(255)-Fx(256-257)-Vb(258)-Fx(259)-Vb(260)-Fx        (261)-Vb(262)-Vb(263)-Vb(264)-Vb(265)-Vb(266)-Vb(267)-Vb(268)-Vb(269)-Vb        (270)-Vb(271)-Vb(272)-Vb(273)-Vb(274)-Vb(275)-Vb(276)-Fx(277)-Vb(278)-Fx        (279)-Vb(280)-Vb(281)-Vb(282)-Vb(283)-Vb(284)-Vb(285)-Vb(286)-Fx(287)-Vb        (288)-Fx(289)-Vb(290)-Vb(291)-Vb(292)-Vb(293)-Vb(294)-Vb(295)-Vb(296)-Vb        (297)-Vb(298)-Vb(299)-Vb(300)-Vb(301)-Vb(302)-Vb(303)-Vb(304)-Vb(305)-Fx        (306-312)-Vb(313)-Fx(314-316)-Vb(317)-Vb(318)-Fx(319)-Vb(320)-Fx(321)-Vb        (322)-Vb(323)-Vb(324)-Vb(325)-Vb(326)-Vb(327)-Vb(328)-Vb(329)-Vb(330)-Vb        (331)-Vb(332)-Vb(333)-Vb(334)-Vb(335)-Vb(336)-Vb(337);    -   wherein Vb(221) is selected from the group consisting of D, K        and Y;    -   Vb(222) is selected from the group consisting of K, E and Y;    -   Vb(223) is selected from the group consisting of T, E and K;    -   Vb(224) is selected from the group consisting of H, E and Y;    -   Vb(225) is selected from the group consisting of T, E, K and W;    -   Fx(226) is C;    -   Vb(227) is selected from the group consisting of P, E, G, K, Y    -   Vb(228) is selected from the group consisting of P, E, G, K, Y    -   Fx(229) is C;    -   Vb(230) is selected from the group consisting of P, A, E, G AND        Y;    -   Vb(231) is selected from the group consisting of A, E, G, K, P        AND Y;    -   Vb(232) is selected from the group consisting of P, E, G, K AND        Y;    -   Vb(233) is selected from the group consisting of A, D, F, G, H,        I, K, L, M, N, Q, R, S, T, V, W, Y;    -   Vb(234) is selected from the group consisting of L, A, D, E, F,        G, H, I, K, M, N, P, Q, R, S, T, V, W, Y    -   Vb(235) is selected from the group consisting of L, A, D, E, F,        G, H, I, K, M, N, P, Q, R, S, T, V, W, Y;    -   Vb(236) is selected from the group consisting of S, A, D, E, F,        H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;    -   Vb(237) is selected from the group consisting of G, D, E, F, H,        I, K, L, M, N, P, Q, R, S, T, V, W, Y;    -   Vb(238) is selected from the group consisting of P, D, E, F, G,        H, I, K, L, M, N, Q, R, S, T, V, W, Y;    -   Vb(239) is selected from the group consisting of S, D, E, F, G,        H, I, K, L, M, N, P, Q, R, T, V, W, Y    -   Vb(240) is selected from the group consisting of V, A, I, M, T;    -   Vb(241) is selected from the group consisting of F, D, E, L, R,        S, W, Y    -   Fx(242) is L;    -   Vb(243) is selected from the group consisting of F, E, H, L, Q,        R, W, Y;    -   Vb(244) is selected from the group consisting of P, H;    -   Vb(245) is selected from the group consisting of P, A;    -   Vb(246) is selected from the group consisting of K, D, E, H, Y;    -   Vb(247) is selected from the group consisting of P, G, V    -   Fx(248) is K;    -   Vb(249) is selected from the group consisting of D, H, O, Y;    -   Fx(250-254) is the sequence -(T-L-M-I-S)-(SEQ ID NO:166)    -   Vb(255) is selected from the group consisting of R, E, Y;    -   Fx(256-257) is the sequence -(T-P)-;    -   Vb(258) is selected from the group consisting of F, H, S, Y;    -   Fx(259) is V;    -   Vb(260) is selected from the group consisting of T, D, E, H, Y;    -   Fx(261) is C;    -   Vb(262) is selected from the group consisting of V, A, E, F, I,        T;    -   Vb(263) is selected from the group consisting of V, A, I, M, T;    -   Vb(264) is selected from the group consisting of V, A, D, E, F,        G, H, I, K, L, M, N, P, Q, R, S, T, W and Y;    -   Vb(265) is selected from the group consisting of D, F, G, H, I,        K, L, M, N, P, Q, R, S, T, V, W, Y;    -   Vb(266) is selected from the group consisting of V, A, I, M, T;    -   Vb(267) is selected from the group consisting of S, D, E, F, H,        I, K, L, M, N, P, Q, R, T, V, W, Y;    -   Vb(268) is selected from the group consisting of H, D, E, F, G,        I, K, L, M, N, P, Q, R, T, V, W, Y;    -   Vb(269) is selected from the group consisting of E, F, G, H, I,        K, L, M, N, P, R, S, T, V, W, Y;    -   Vb(270) is selected from the group consisting of D, F, G, H, I,        L, M, P, Q, R, S, T, W, Y;    -   Vb(271) is selected from the group consisting of A, D, E, F, G,        H, I, K, L, M, N, Q, R, S, T, V, W, Y;    -   Vb(272) is selected from the group consisting of E, D, F, G, H,        I, K, L, M, P, R, S, T, V, W, Y;    -   Vb(273) is selected from the group consisting of V, I;    -   Vb(274) is selected from the group consisting of K, D, E, F, G,        H, L, M, N, P, R, T, V, W, Y;    -   Vb(275) is selected from the group consisting of F, L, W;    -   Vb(276) is selected from the group consisting of N, D, E, F, G,        H, I, L, M, P, R, S, T, V, W, Y;    -   Fx(277) is W;    -   Vb(278) is selected from the group consisting of Y, D, E, G, H,        I, K, L, M, N, P, Q, R, S, T, V, W;    -   Fx(279) is V;    -   Vb(280) is selected from the group consisting of D, G, K, L, P,        W;    -   Vb(281) is selected from the group consisting of G, D, E, K, N,        P, Q, Y;    -   Vb(282) is selected from the group consisting of V, E, G, K, P,        Y;    -   Vb(283) is selected from the group consisting of E, G, H, K, L,        P, R, Y;    -   Vb(284) is selected from the group consisting of V, D, E, L, N,        Q, T, Y;    -   Vb(285) is selected from the group consisting of H, D, E, K, Q,        W, Y;    -   Vb(286) is selected from the group consisting of N, E, G, P, Y;    -   Fx(287) is selected from the group consisting of A;    -   Vb(288) is selected from the group consisting of K, D, E, Y;    -   Fx(289) is T;    -   Vb(290) is selected from the group consisting of K, D, H, L, N,        W;    -   Vb(291) is selected from the group consisting of P, D, E, G, H,        I, Q, T;    -   Vb(292) is selected from the group consisting of R, D, E, T, Y;    -   Vb(293) is selected from the group consisting of E, F, G, H, I,        L, M, N, P, R, S, T, V, W, Y;    -   Vb(294) is selected from the group consisting of E, F, G, H, I,        K, L, M, P, R, S, T, V, W, Y;    -   Vb(295) is selected from the group consisting of Q, D, E, F, G,        H, I, M, N, P, R, S, T, V, W, Y;    -   Vb(296) is selected from the group consisting of Y, A, D, E, G,        H, I, K, L, M, N, Q, R, S, T, V;    -   Vb(297) is selected from the group consisting of N, D, E, F, G,        H, I, K, L, M, P, Q, R, S, T, V, W, Y;    -   Vb(298) is selected from the group consisting of S, D, E, F, H,        I, K, M, N, Q, R, T, W, Y;    -   Vb(299) is selected from the group consisting of T, A, D, E, F,        G, H, I, K, L, M, N, P, Q, R, S, V, W, Y;    -   Vb(300) is selected from the group consisting of Y, A, D, E, G,        H, K, M, N, P, Q, R, S, T, V, W;    -   Vb(301) is selected from the group consisting of R, D, E, H, Y;    -   Vb(302) is selected from the group consisting of V, I;    -   Vb(303) is selected from the group consisting of V, D, E, Y;    -   Vb(304) is selected from the group consisting of S, D, H, L, N,        T;    -   Vb(305) is selected from the group consisting of V, E, T, Y,    -   Fx(306-312) is -(L-T-V-L-H-Q-D)-(SEQ ID NO:167);    -   Vb(313) is selected from the group consisting of W, F;    -   Fx(314-316) is -(L-N-G)-;    -   Vb(317) is selected from the group consisting of K, E, Q;    -   Vb(318) is selected from the group consisting of E, H, L, Q, R,        Y;    -   Fx(319) is Y;    -   Vb(320) is selected from the group consisting of K, D, F, G, H,        I, L, N, P, S, T, V, W, Y;    -   Fx(321) is C;    -   Vb(322) is selected from the group consisting of K, D, F, G, H,        I, P, S, T, V, W, Y;    -   Vb(323) is selected from the group consisting of V, I;    -   Vb(324) is selected from the group consisting of S, D, F, G, H,        I, L, M, P, R, T, V, W, Y;    -   Vb(325) is selected from the group consisting of N, A, D, E, F,        G, H, I, K, L, M, P, Q, R, S, T, V, W, Y;    -   Vb(326) is selected from the group consisting of K, I, L, P, T;    -   Vb(327) is selected from the group consisting of A ,D, E, F, H,        I, K, L, M, N, P, R, S, T, V, W, Y;    -   Vb(328) is selected from the group consisting of L, A, D, E, F,        G, H, I, K, M, N, P, Q, R, S, T, V, W, Y;    -   Vb(329) is selected from the group consisting of P, D, E, F, G,        H, I, K, L, M, N, Q, R, S, T, V, W, Y;    -   Vb(330) is selected from the group consisting of A, E, F, G, H,        I, L, M, N, P, R, S, T, V, W, Y;    -   Vb(331) is selected from the group consisting of P, D, F, H, I,        L, M, Q, R, T, V, W, Y;    -   Vb(332) is selected from the group consisting of I, A, D, E, F,        H, K, L, M, N, P, Q, R, S, T, V, W, Y;    -   Vb(333) is selected from the group consisting of E, F, H, I, L,        M, N, P, T, Y;    -   Vb(334) is selected from the group consisting of K, F, I, L, P,        T;    -   Vb(335) is selected from the group consisting of T, D, F, G, H,        I, L, M, N, P, R, S, V, W, Y;    -   Vb(336) is selected from the group consisting of I, E, K, Y;    -   Vb(337) is selected from the group consisting of S, E, H, N.

In another embodiment, the Fc region comprises is a variant selectedfrom the group consisting of: S239D/I332E, S239D/G236A, S239D/G236S,S239D/V264I, S239D/H268D, S239D/H268E, S239D/S298A, S239D/K326E,S239D/A330L, S239D/A330Y, S239D/A330I, I332E/V264I, I332E/H268D,I332E/H268E, I332E/S298A, I332E/K326E, I332E/A330L, I332E/A330Y,I332E/A330I, I332E/G236A, I332E/G236A, I332D/V264I, I332D/H268D,I332D/H268E, I332D/S298A, I332D/K326E, I332D/A330L, I332D/A330Y,I332D/A330I, I332D/G236A, I332D/G236S, S239D/K246H/I332E,S239D/V264I/I332E, S239D/S267E/I332E, S239D/H268D/I332E,S239D/H268E/I332E, S239D/S298A/I332E, S239D/S324G/I332E,S239D/S324I/I332E, S239D/K326T/I332E, S239D/K326E/I332E,S239D/K326D/I332E, S239D/A327D/I332E, S239D/A330L/I332E,S239D/A330Y/I332E, S239D/A330I/I332E, S239D/K334T/I332E,S239D/K246H/T260H/I332E, S239D/K246H/H258D/I332E,S239D/K246H/H268E/I332E, S239D/H268D/S324G/I332E,S239D/H268E/S324G/I332E, S239D/H268D/K326T/I332E,S239D/H268E/K326T/I332E, S239D/H268D/A330L/I332E,S239D/H268E/A330L/I332E, S239D/H268D/A330Y/I332E,S239D/H268E/A330Y/I332E, S239D/S298A/S267E/I332E,S239D/S298A/H268D/I332E, S239D/S298A/H268E/I332E,S239D/S298A/S324G/I332E, S239D/S298A/S324I/I332E,S239D/S298A/K326T/I332E, S239D/S298A/K326E/I332E,S239D/S298A/A327D/I332E, S239D/S298A/A330L/I332E,S239D/S298A/A330Y/I332E, S239D/K326T/A330Y/I332E,S239D/K326E/A330Y/I332E, S239D/K326T/A330L/I332E, andS239D/K326E/A330L/I332E, wherein numbering is according to the EU index.

In a further variation, the Fc variant is selected from the groupconsisting of: G236S, G236A, S239D, S239E, S239N, S239Q, S239T, K246H,T260H, K246Y, D249Y, R255Y, E258Y, V264I, S267E, H268D, H268E, E272Y,E272I, E272H, K274E, G281D, E283L, E283H, S304T, S324G, S3241, K326T,A327D, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, E333Y, K334T,and K334F, wherein numbering is according to the EU index.

In another variation, the Fc variant further comprising a substitutionselected from the group consisting of S298A, K326E, K326D, K326A, E333A,and K334A, wherein numbering is according to the EU index. In stillother variations, the Fc variant comprising at least one amino acidmodification in the Fc region of said parent Fc polypeptide, whereinsaid variant protein selectively enhances binding to one or more Fcligands relative to one or more other Fc ligands, and wherein said Fcvariant comprises a substitution at a position selected from the groupconsisting of: 234, 235, 236, 267, 268, 292, 293, 295, 300, 324, 327,328, 330, and 335, wherein numbering is according to the EU index.

Preferred embodiments comprise optimization of Fc binding to a humanFcγR, however in alternate embodiments the Ep-CAM targeting proteins ofthe present invention possess enhanced or reduced affinity for FcγRsfrom nonhuman organisms, including but not limited to rodents andnon-human primates. Ep-CAM targeting proteins that are optimized forbinding to a nonhuman FcγR may find use in experimentation. For example,mouse models are available for a variety of diseases that enable testingof properties such as efficacy, toxicity, and pharmacokinetics for agiven drug candidate. As is known in the art, cancer cells can begrafted or injected into mice to mimic a human cancer, a processreferred to as xenografting. Testing of Ep-CAM targeting proteins thatcomprise Ep-CAM targeting proteins that are optimized for one or moremouse FcγRs, may provide valuable information with regard to theefficacy of the protein, its mechanism of action, and the like. TheEPCAM targeting proteins of the present invention may also be optimizedfor enhanced functionality and/or solution properties in aglycosylatedform. In a preferred embodiment, the aglycosylated Ep-CAM targetingproteins of the present invention bind an Fc ligand with greateraffinity than the aglycosylated form of the parent Ep-CAM targetingprotein. The Fc ligands include but are not limited to FcγRs, C1q, FcRn,and proteins A and G, and may be from any source including but notlimited to human, mouse, rat, rabbit, or monkey, preferably human. In analternately preferred embodiment, the Ep-CAM targeting proteins areoptimized to be more stable and/or more soluble than the aglycosylatedform of the parent Ep-CAM targeting protein.

Ep-CAM targeting proteins of the invention may comprise modificationsthat modulate interaction with Fc ligands other than FcγRs, includingbut not limited to complement proteins, FcRn, and Fc receptor homologs(FcRHs). FcRHs include but are not limited to FcRH1, FcRH2, FcRH3,FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews190:123-136, expressly incorporated by reference). The modificationsthat modulate FcRn binding may be used to increase or decrease the invivo half-life of the EP-CAM targeting protein. Decreasing the in vivohalf-life is useful in decreasing the toxicity of a therapeutic Ep-CAMtargeting protein or in in vivo imaging procedures. More preferably,increasing the in vivo half-life is useful to increase the potency of atherapeutic Ep-CAM targeting protein and may be done by increasing theFcRn binding of the Ep-CAM targeting protein at slightly acidic pH(typically about pH 6.0). (See Burmeister et al. 1994 Nature372:336-343; Israel et al. 1996 Immunology 89(4):573-578; Junghans andAnderson 1996 Proc. Natl. Acad. Sci. USA 93:5512-5516; Ghetie et al.1996 Eur J. Immunol. 26:690-696. Hinton et al. 2004 J. Biol. Chem.279(8):6213-6216; US06277375; and U.S. Ser. No. 10/822,300; allexpressly incorporated by reference).

Preferably, the Fc ligand specificity of the Ep-CAM targeting protein ofthe present invention will determine its therapeutic utility. Theutility of a given Ep-CAM targeting protein for therapeutic purposeswill depend on the epitope or form of the Ep-CAM target antigen and thedisease or indication being treated. For some targets and indications,enhanced FcγR-mediated effector functions may be preferable. This may beparticularly favorable for anti-cancer Ep-CAM targeting proteins. ThusEp-CAM targeting proteins may be used that comprise Ep-CAM targetingproteins that provide enhanced affinity for activating FcγRs and/orreduced affinity for inhibitory FcγRs. For some targets and indications,it may be further beneficial to utilize Ep-CAM targeting proteins thatprovide differential selectivity for different activating FcγRs; forexample, in some cases enhanced binding to FcγRIIa and FcγRIIIa may bedesired, but not FcγRI, whereas in other cases, enhanced binding only toFcγRIIa may be preferred. For certain targets and indications, it may bepreferable to utilize Ep-CAM targeting proteins that enhance bothFcγR-mediated and complement-mediated effector functions, whereas forother cases it may be advantageous to utilize Ep-CAM targeting proteinsthat enhance either FcγR-mediated or complement-mediated effectorfunctions. For some Ep-CAM targets or cancer indications, it may beadvantageous to reduce or ablate one or more effector functions, forexample by knocking out binding to C1q, one or more FcγR's, FcRn, or oneor more other Fc ligands. For other targets and indications, it may bepreferable to utilize Ep-CAM targeting proteins that provide enhancedbinding to the inhibitory FcγRIIb, yet WT level, reduced, or ablatedbinding to activating FcγRs. This may be particularly useful, forexample, when the goal of an Ep-CAM targeting protein is to inhibitinflammation or auto-immune disease, or modulate the immune system insome way.

Clearly an important parameter that determines the most beneficialselectivity of a given Ep-CAM targeting protein to treat a given diseaseis the context of the Ep-CAM targeting protein, that is, what type ofEp-CAM targeting protein is being used. Thus the Fc ligand selectivityor specifity of a given Ep-CAM targeting protein will provide differentproperties depending on whether it composes an antibody, Fc fusion, oran Ep-CAM targeting protein with a coupled fusion or conjugate partner.For example, toxin, radionucleotide, or other conjugates may be lesstoxic to normal cells if the Ep-CAM targeting protein that comprisesthem has reduced or ablated binding to one or more Fc ligands. Asanother example, in order to inhibit inflammation or auto-immunedisease, it may be preferable to utilize an Ep-CAM targeting proteinwith enhanced affinity for activating FcγRs, such as to bind these FcγRsand prevent their activation. Conversely, an Ep-CAM targeting proteinthat comprises two or more Fc regions with enhanced FcγRIIb affinity mayco-engage this receptor on the surface of immune cells, therebyinhibiting proliferation of these cells. Whereas in some cases an Ep-CAMtargeting protein may engage its target antigen on one cell type yetengage FcγRs on separate cells from the target antigen, in other casesit may be advantageous to engage FcγRs on the surface of the same cellsas the target antigen. For example, if an antibody targets an antigen ona cell that also expresses one or more FcγRs, it may be beneficial toutilize an Ep-CAM targeting protein that enhances or reduces binding tothe FcγRs on the surface of that cell. This may be the case, for examplewhen the Ep-CAM targeting protein is being used as an anti-cancer agent,and co-engagement of target antigen and FcγR on the surface of the samecell promote signalling events within the cell that result in growthinhibition, apoptosis, or other anti-proliferative effect.Alternatively, antigen and FcγR co-engagement on the same cell may beadvantageous when the Ep-CAM targeting protein is being used to modulatethe immune system in some way, wherein co-engagement of target antigenand FcγR provides some proliferative or anti-proliferative effect.Likewise, Ep-CAM targeting proteins that comprise two or more Fc regionsmay benefit from Ep-CAM targeting proteins that modulate FcγRselectivity or specifity to co-engage FcγRs on the surface of the samecell.

The Fc ligand specificity of the Ep-CAM targeting proteins of thepresent invention can be modulated to create different effector functionprofiles that may be suited for particular Ep-CAM epitopes, indicationsor patient populations. Table 1 describes several preferred embodimentsof receptor binding profiles that include improvements to, reductions toor no effect to the binding to various receptors, where such changes maybe beneficial in certain contexts. The receptor binding profiles in thetable could be varied by degree of increase or decrease to the specifiedreceptors. Additionally, the binding changes specified could be in thecontext of additional binding changes to other receptors such as C1q orFcRn, for example by combining with ablation of binding to C1q to shutoff complement activation, or by combining with enhanced binding to C1qto increase complement activation. Other embodiments with other receptorbinding profiles are possible, the listed receptor binding profiles areexemplary. TABLE 1 Receptor Receptor binding binding Therapeuticimprovement reduction Cell activity activity Solely I — enhancedendritic cell activity Enhancement and uptake, and subsequencecell-based presentation of immune antigens; enhance monocyte responseand macrophage response to against antibody target IIIa Enhance ADCC andIncreased phagocytosis of broad range target cell of cell types lysisIIIa IIb Enhance ADCC and Increased phagocytosis of broad range targetcell of cell types lysis IIb, Iic Reduction of activity of allEnhancement FcR bearing cell types except of target NK cells andpossible cell lysis activation of NK cells via IIc selective receptorsignaling for NK cell accessible target cells IIb, IIIa — Possible NKcell specific Enhancement activation and enhancement of target of NKcell mediated ADCC cell lysis selective for NK cell accessible targetcells IIIb Neutrophil mediated Enhanced phagocytosis enhancement targetcell destruction for neutrophil accessible cells FcαR Neutrophilmediated Enhanced phagocytosis enhancement target cell destruction forneutrophil accessible cells I, IIa, IIIa IIb enhance dendritic cellactivity enhance and uptake, and subsequence cell-based presentation ofantigens to T immune cells; enhance monocyte and response macrophageresponse to against antibody target IIb IIIa, IIa, I Reduction inactivity of Eliminate or monocytes, macrophages, reduce neutrophils, NK,dendritic and cell-mediated other gamma receptor cytotoxicity bearingcells against target bearing cells

The presence of different polymorphic forms of FcγRs provides yetanother parameter that impacts the therapeutic utility of the Ep-CAMtargeting proteins of the present invention. Whereas the specificity andselectivity of a given Ep-CAM targeting protein for the differentclasses of FcγRs signficantly affects the capacity of an Ep-CAMtargeting protein to target a given antigen for treatment of a givendisease, the specificity or selectivity of an Ep-CAM targeting proteinfor different polymorphic forms of these receptors may in part determinewhich research or pre-clinical experiments may be appropriate fortesting, and ultimately which patient populations may or may not respondto treatment. Thus the specificity or selectivity of Ep-CAM targetingproteins of the present invention to Fc ligand polymorphisms, includingbut not limited to FcγR, C1q, FcRn, and FcRH polymorphisms, may be usedto guide the selection of valid research and pre-clinical experiments,clinical trial design, patient selection, dosing dependence, and/orother aspects concerning clinical trials.

The Ep-CAM targeting proteins of the present invention may be combinedwith other amino acid modifications in the Fc region that providealtered or optimized interaction with one or more Fc ligands, includingbut not limited to FcγRs, C1q, FcRn, FcR homologues, and/or as yetundiscovered Fc ligands. Additional modifications may provide altered oroptimized affinity and/or specificity to the Fc ligands. Additionalmodifications may provide altered or optimized effector functions,including but not limited to ADCC, ADCP, CDC, and/or serum half-life.Such combination may provide additive, synergistic, or novel propertiesin antibodies or Fc fusions. In one embodiment, the Ep-CAM targetingproteins of the present invention may be combined with known Fc variants(Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J Immunol147:2657-2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al.,1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc NatlAcad Sci U S A 92:11980-11984; Jefferis et al., 1995, Immunol Lett44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996,Immunol Lett 54:101-104; Lund et al., 1996, J Immunol 157:4963-4969;Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000,J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xuet al., 2000, Cell Immunol 200:16-26; Idusogie et al., 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490; Hinton et al., 2004, J Biol Chem 279:6213-6216; U.S.Pat. Nos. 5,624,821; 5,885,573; 6,194,551; PCT WO 00/42072; PCT WO99/58572; US 2004/0002587 A1), U.S. Pat. No. 6,737,056, PCTUS2004/000643, U.S. Ser. No. 10/370,749, and PCT/US2004/005112; allexpressly incorporated by reference). For example, as described in U.S.Pat. 6,737,056, PCT US2004/000643, U.S. Ser. No. 10/370,749, andPCT/US2004/005112, the substitutions S298A, S298D, K326E, K326D, E333A,K334A, and P396L provide optimized FcγR binding and/or enhanced ADCC.Furthermore, as disclosed in Idusogie et al., 2001, J. Immunology166:2571-2572, substitutions K326W, K326Y, and E333S provide enhancedbinding to the complement protein C1q and enhanced CDC. Finally, asdescribed in Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216,substitutions T250Q, T250E, M428L, and M428F provide enhanced binding toFcRn and improved pharmacokinetics.

Because the binding sites for FcγRs, C1q, and FcRn reside in the Fcregion, the differences between the IgGs in the Fc region are likely tocontribute to differences in FcγR- and C1q-mediated effector functions.It is also possible that the modifications can be made in other non-Fcregions of an Ep-CAM targeting protein, including for example the Faband hinge regions of an antibody, or the Fc fusion partner of an Fcfusion. For example, as disclosed in U.S. Ser. No. 60/614,944; U.S. Ser.No. 60/585,328; U.S. Ser. No. 60/573,302; entitled “ImmunoglobulinVariants Outside the Fc Region with Optimized Effector Function”, theFab and hinge regions of an antibody may impact effector functions suchas antibody dependent cell-mediated cytotoxicity (ADCC), antibodydependent cell-mediated phagocytosis (ADCP), and complement dependentcytotoxicity (CDC). Thus modifications outside the Fc region of anEp-CAM targeting protein of the present invention are contemplated. Forexample, anti-Ep-CAM antibodies of the present invention may compriseone or more amino acid modifications in the VL, CL, VH, CH1, and/orhinge regions of an antibody.

Other modifications may provide additional or novel binding determinantsinto an Ep-CAM targeting protein, for example additional or novel Fcreceptor binding sites, for example as described in U.S. Ser. No.60/531,752, filed Dec. 22, 2003, entitled “Ep-CAM targeting proteinswith novel Fc receptor binding sites”. In one embodiment, an Ep-CAMtargeting protein of one antibody isotype may be engineered such that itbinds to an Fc receptor of a different isotype. This may be particularlyapplicable when the Fc binding sites for the respective Fc receptors donot significantly overlap. For example, the structural determinants ofIgA binding to FcγRI may be engineered into an IgG Ep-CAM targetingprotein.

The Ep-CAM targeting proteins of the present invention may comprisemodifications that modulate the in vivo pharmacokinetic properties of anEp-CAM targeting protein. These include, but are not limited to,modifications that enhance affinity for the neonatal Fc receptor FcRn(U.S. Ser. No. 10/020354; WO2001US0048432; EP 2001000997063; U.S. Pat.No. 6,277,375; U.S. Ser. No. 09/933497; WO1997US0003321; U.S. Pat. No.6,737,056; WO2000US0000973; Shields et al. J. Biol. Chem., 276(9),6591-6604 (2001); Zhou et al. J. Mol. Biol., 332, 901-913 (2003), allexpressly incorporated by reference). These further includemodifications that modify FcRn affinity in a pH-specific manner. In someembodiments, where enhanced in vivo half-life is desired, modificationsthat specifically enhance FcRn affinity at lower pH (5.5-6) relative tohigher pH (7-8) are preferred (Hinton et al. J. Biol. Chem. 279(8),6213-6216 (2004); Dall' Acqua et al. J. Immuno. 169, 5171-5180 (2002);Ghetie et al. Nat. Biotechnol., 15(7), 637-640 (1997), WO2003US0033037;WO2004US0011213, all expressly incorporated by reference). For example,as described in Hinton et al., 2004, “Engineered Human IgG Antibodieswith Longer Serum Half-lives in Primates” J. Biol. Chem. 279(8):6213-6216, substitutions T250Q, T250E, M428L, and M428F provide enhancedbinding to FcRn and improved pharmacokinetics. Additionally preferredmodifications are those that maintain the wild-type Fc's improvedbinding at lower pH relative to the higher pH. In alternativeembodiments, where rapid in vivo clearance is desired, modificationsthat reduce affinity for FcRn are preferred. (U.S. Pat. No. 6,165,745;WO1993US0003895; EP1993000910800; WO1997US0021437; Medesan et al., J.Immunol., 158(5), 2211-2217 (1997); Ghetie and Ward, Annu. Rev.Immunol., 18, 739-766 (2000); Martin et al. Molecular Cell, 7, 867-877(2001); Kim et al. Eur. J. Immunol. 29, 2819-2825 (1999), all expresslyincorporated by reference).

Ep-CAM targeting proteins of the present invention may comprise one ormore modifications that provide optimized properties that are notspecifically related to effector function per se. The modifications maybe amino acid modifications, or may be modifications that are madeenzymatically or chemically. Such modification(s) likely provide someimprovement in the Ep-CAM targeting protein, for example an enhancementin its stability, solubility, function, or clinical use. The presentinvention contemplates a variety of improvements that made be made bycoupling the Ep-CAM targeting proteins of the present invention withadditional modifications.

In a preferred embodiment, the Ep-CAM targeting proteins of the presentinvention may comprise modifications to reduce immunogenicity in humans.In a most preferred embodiment, the immunogenicity of an Ep-CAMtargeting protein of the present invention is reduced using a methoddescribed in U.S. Ser. No. 60/1619,483, filed Oct. 14, 2004 and U.S.Ser. No. 11/004,590, entitled “Methods of Generating Variant Proteinswith Increased Host String Content and Compositions Thereof”, filed onDec. 6, 2004. In alternate embodiments, the antibodies of the presentinvention are humanized (Clark, 2000, Immunol Today 21:397-402,expressly incorporated by reference). In CDR grafting, humanizationrelies principally on the grafting of donor CDRs onto acceptor (human)VL and VH frameworks (Winter U.S. Pat. No. 5,225,539, expresslyincorporated by reference). “Backmutation” of selected acceptorframework residues to the corresponding donor residues is often requiredto regain affinity that is lost in the initial grafted construct (U.S.Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370;5,859,205; 5,821,337; 6,054,297; 6,407,213, all expressly incorporatedby reference). The humanized antibody optimally also will comprise atleast a portion of an immunoglobulin constant region, typically that ofa human immunoglobulin, and thus will typically comprise a human Fcregion. A variety of techniques and methods for humanizing and reshapingnon-human antibodies are well known in the art (See Tsurushita &Vasquez. 2004, Humanization of Monoclonal Antibodies, Molecular Biologyof B Cells, 533-545, Elsevier Science (USA), expressly incorporated byreference). Humanization methods include but are not limited to methodsdescribed in Jones et al., 1986, Nature 321:522-525; Riechmann et al.,1988; Nature 332:323-329; Verhoeyen et al., 1988, Science,239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33;He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, ProcNatl Acad Sci USA 89:4285-9, Presta et al., 1997, CancerRes.57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA88:41814185; O'Connor et al., 1998, Protein Eng 11:321-8; all expresslyincorporated by reference. Humanization or other methods of reducing theimmunogenicity of nonhuman antibody variable regions may includeresurfacing methods, as described for example in Roguska et al., 1994,Proc. Natl. Acad. Sci. USA 91:969-973. In one embodiment, selectionbased methods may be employed to humanize and/or affinity matureantibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618, Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759; all expressly incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,502; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084; all expressly incorporated byreference. Structure-based methods may be employed for humanization andaffinity maturation, for example as described in U.S. Ser. No.10/153,159, expressly incorporated by reference, and relatedapplications.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an Ep-CAMtargeting protein of the present invention. See for example WO 98152976;WO 02/079232; WO 0013317; U.S. Ser. Nos. 09/903,378; 10/039,170,60/222,697; 10/339788; PCT WO 01/21823; and PCT WO 02/00165; Mallios,1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17:942-948; Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO98/59244; WO 02/069232; WO 02/77187; Marshall et al., 1995, J. Immunol.154: 5927-5933; and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358;all expressly incorporated by reference. Sequence-based information canbe used to determine a binding score for a given peptide—MHC interaction(see for example Mallios, 1999, Bioinformatics 15: 432-439; Mallios,2001, Bioinformatics 17: p 942-948; Sturniolo et. al., 1999, NatureBiotech. 17: 555-561, all expressly incorporated by reference). It ispossible to use structure-based methods in which a given peptide iscomputationally placed in the peptide-binding groove of a given MHCmolecule and the interaction energy is determined (for example, see WO98/59244 and WO 02/069232, both expressly incorporated by reference).Such methods may be referred to as “threading” methods. Alternatively,purely experimental methods can be used; for example a set ofoverlapping peptides derived from the protein of interest can beexperimentally tested for the ability to induce T-cell activation and/orother aspects of an immune response. (see for example WO 02/77187,expressly incorporated by reference). In a preferred embodiment,MHC-binding propensity scores are calculated for each 9-residue framealong the protein sequence using a matrix method (see Sturniolo et. al.,supra; Marshall et. al., 1995, J. Immunol. 154: 5927-5933, and Hammer etal., 1994, J. Exp. Med. 180: 2353-2358; all expressly incorporated byreference). It is also possible to consider scores for only a subset ofthese residues, or to consider also the identities of the peptideresidues before and after the 9-residue frame of interest. The matrixcomprises binding scores for specific amino acids interacting with thepeptide binding pockets in different human class II MHC molecule. In themost preferred embodiment, the scores in the matrix are obtained fromexperimental peptide binding studies, In an alternate preferredembodiment, scores for a given amino acid binding to a given pocket areextrapolated from experimentally characterized alleles to additionalalleles with identical or similar residues lining that pocket. Matricesthat are produced by extrapolation are referred to as “virtualmatrices”. In an alternate embodiment, additional amino acidmodifications may be engineered to reduce the propensity of the intactmolecule to interact with B cell receptors and circulating antibodies.

Anti-Ep-CAM antibodies and Fc fusions of the present invention maycomprise amino acid modifications in one or more regions outside the Fcregion, for example the antibody Fab region or the Fc fusion partner,that provide optimal properties. In one embodiment, the variable regionof an antibody of the present invention may be affinity matured, that isto say that amino acid modifications have been made in the VH and/or VLdomains of the antibody to enhance binding of the antibody to its targetantigen. Likewise, modifications may be made in the Fc fusion partner toenhance affinity of the Fc fusion for its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Other improvements tothe target recognition properties may be provided by additionalmodifications. Such properties may include, but are not limited to,specific kinetic properties (i.e. association and dissociationkinetics), selectivity for the particular target versus alternativetargets, and selectivity for a specific form of target versusalternative forms. Examples include full-length versus splice variants,cell-surface vs. soluble forms, selectivity for various polymorphicvariants, or selectivity for specific conformational forms of the Ep-CAMtarget.

Ep-CAM targeting proteins of the invention may comprise one or moremodifications that provide reduced or enhanced internalization of anEp-CAM targeting protein. In one embodiment, Ep-CAM targeting proteinsof the present invention can be utilized or combined with additionalmodifications in order to reduce the cellular internalization of anEp-CAM targeting protein that occurs via interaction with one or more Fcligands. This property might be expected to enhance effector function,and potentially reduce immunogenicity of the Ep-CAM targeting proteinsof the invention. Alternatively, Ep-CAM targeting proteins of thepresent invention can be utilized directly or combined with additionalmodifications in order to enhance the cellular internalization of anEp-CAM targeting protein that occurs via interaction with one or more Fcligands. For example, in a prefered embodiment, an Ep-CAM targetingprotein is used that provides enhanced binding to FcγRI, which isexpressed on dendritic cells and active early in immune response, Thisstrategy could be further enhanced by combination with additionalmodifications, either within the Ep-CAM targeting protein or in anattached fusion or conjugate partner, that promote recognition andpresentation of Fc peptide fragments by MHC molecules. These strategiesare expected to enhance target antigen processing and thereby improveantigenicity of the target antigen (Bonnerot and Amigorena, 1999,Immunol Rev. 172:279-84, expressly incorporated by reference), promotingan adaptive immune response and greater target cell killing by the humanimmune system. These strategies may be particularly advantageous whenthe targeted antigen is shed from the cellular surface. An additionalapplication of these concepts arises with idiotype vaccineimmunotherapies, in which clone-specific antibodies produced by apatient's lymphoma cells are used to vaccinate the patient.

In a preferred embodiment, modifications are made to improve biophysicalproperties of the Ep-CAM targeting proteins of the present invention,including but not limited to stability, solubility, and oligomericstate. Modifications can include, for example, substitutions thatprovide more favorable intramolecular interactions in the Ep-CAMtargeting protein such as to provide greater stability, or substitutionof exposed nonpolar amino acids with polar amino acids for highersolubility. A number of optimization goals and methods are described inU.S. Ser. No. 10/379,392, expressly incorporated by reference, that mayfind use for engineering additional modifications to further optimizethe Ep-CAM targeting proteins of the present invention. The Ep-CAMtargeting proteins of the present invention can also be combined withadditional modifications that reduce oligomeric state or size, such thattumor penetration is enhanced, or in vivo clearance rates are increasedas desired.

Other modifications to the Ep-CAM targeting proteins of the presentinvention include those that enable the specific formation orhomodimeric or homomultimeric molecules. Such modifications include butare not limited to engineered disulfides, as well as chemicalmodifications or aggregation methods, which may provide a mechanism forgenerating covalent homodimeric or homomultimers. For example, methodsof engineering and compositions of such molecules are described in Kanet al., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002,Recent Results Cancer Res. 159: 104-12; U.S. Pat. No. 5,681,566; Caronet al., 1992, J. Exp. Med. 176:1191-1195, and Shopes, 1992, J. Immunol.148(9):2918-22; all expressly incorporated by reference. Additionalmodifications to the variants of the present invention include thosethat enable the specific formation or heterodimeric, heteromultimeric,bifunctional, and/or multifunctional molecules. Such modificationsinclude, but are not limited to, one or more amino acid substitutions inthe CH3 domain, in which the substitutions reduce homodimer formationand increase heterodimer formation. For example, methods of engineeringand compositions of such molecules are described in Atwell et al., 1997,J. Mol. Biol. 270(1):26-35, and Carter et al., 2001, J. Immunol. Methods248:7-15; both expressly incorporated by reference. Additionalmodifications include modifications in the hinge and CH3 domains, inwhich the modifications reduce the propensity to form dimers.

In further embodiments, the Ep-CAM targeting proteins of the presentinvention comprise modifications that remove proteolytic degradationsites. These may include, for example, protease sites that reduceproduction yields, as well as protease sites that degrade theadministered protein in vivo. In a preferred embodiment, additionalmodifications are made to remove covalent degradation sites such asdeamidation (i.e. deamidation of glutaminyl and asparaginyl residues tothe corresponding glutamyl and aspartyl residues), oxidation, andproteolytic degradation sites. Deamidation sites that are particularuseful to remove are those that have enhance propensity for deamidation,including, but not limited to asparaginyl and gituamyl residues followedby glycines (NG and QG motifs, respectively). In such cases,substitution of either residue can significantly reduce the tendency fordeamidation. Common oxidation sites include methionine and cysteineresidues. Other covalent modifications, that can either be introduced orremoved, include hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the aminogroups of lysine, arginine, and histidine side chains [T. E. Creighton,Proteins, Structure and Molecular Properties, W.H. Freeman & Co., SanFrancisco, pp. 79-86 (1983), expressly incorporated by reference],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. Additional modifications also may include but are notlimited to posttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression andlorpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO), yeast cell lines, bacterial cell lines,and plants. Additional modifications include modifications that removeor reduce the ability of heavy chains to form inter-chain disulfidelinkages. Additional modifications include modifications that remove orreduce the ability of heavy chains to form intra-chain disulfidelinkages.

The Ep-CAM targeting proteins of the present invention may comprisemodifications that include the use of unnatural amino acids incorporatedusing, for example, the technologies developed by Schultz andcolleagues, including but not limited to methods described by Cropp &Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc.Natl. Acad. Sci. U.S.A. 101(2):7566-71, Zhang et al., 2003,303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7;expressly incorporated by reference. In some embodiments, thesemodifications enable manipulation of various functional, biophysical,immunological, or manufacturing properties discussed above. Inadditional embodiments, these modifications enable additional chemicalmodification for other purposes. Other modifications are contemplatedherein. For example, the Ep-CAM targeting protein may be linked to oneof a variety of nonproteinaceous polymers, e.g., polyethylene glycol(PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol. Additional amino acidmodifications may be made to enable specific or non-specific chemical orposttranslational modification of the Ep-CAM targeting proteins. Suchmodifications, include, but are not limited to PEGylation andglycosylation. Specific substitutions that can be utilized to enablePEGylation include, but are not limited to, introduction of novelcysteine residues or unnatural amino acids such that efficient andspecific coupling chemistries can be used to attach a PEG or otherwisepolymeric moiety. Introduction of specific glycosylation sites can beachieved by introducing novel N-X-T/S sequences into the Ep-CAMtargeting proteins of the present invention.

In one embodiment, the Ep-CAM targeting proteins of the presentinvention comprise one or more engineered glycoforms. By “engineeredglycoform” as used herein is meant a carbohydrate composition that iscovalently attached to an Ep-CAM targeting protein, wherein thecarbohydrate composition differs chemically from that of a parent Ep-CAMtargeting protein. An engineered protein comprising a position thatlacks an attached oligosaccharide or carbohydrate may be referred to ascomprising an engineered glycoform, if its parent molecule comprises anoligosaccharide, or carbohydrate at that position. Engineered glycoformsmay be useful for a variety of purposes, including but not limited toenhancing or reducing effector function. Engineered glycoforms may begenerated by a variety of methods known in the art (Umaña et al., 1999,Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawaet al., 2003, J Biol Chem 278:3466-3473; U.S. Pat. No. 6,602,684; U.S.Ser. Nos. 10/277,370; 10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1;PCT WO 02/31140A1; PCT WO 02/30954A1; all expressly incorporated byreference; Potelligent™ technology [Biowa, Inc., Princeton, N.J.];GlycoMAb™ glycosylation engineering technology [GLYCART biotechnologyAG, Zürich, Switzerland]). Many of these techniques are based oncontrolling the level of fucosylated and/or bisecting oligosaccharidesthat are covalently attached to the Fc region, for example by expressingan Ep-CAM targeting protein in various organisms or cell lines,engineered or otherwise (for example Lec-13 CHO cells or rat hybridomaYB2/0 cells), by regulating enzymes involved in the glycosylationpathway (for example FUT8 [α1,6-fucosyltranserase] and/orβ1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by modifyingcarbohydrate(s) after the Ep-CAM targeting protein has been expressed.Engineered glycoform typically refers to the different carbohydrate oroligosaccharide; thus an Ep-CAM targeting protein, for example ananti-Ep-CAM antibody or Fc fusion, may comprise an engineered glycoform.Alternatively, engineered glycoform may refer to the Ep-CAM targetingprotein that comprises the different carbohydrate or oligosaccharide.

The Ep-CAM targeting proteins of the present invention may be fused orconjugated to one or more other molecules or polypeptides. Conjugate andfusion partners may be any molecule, including small molecule chemicalcompounds and polypeptides. For example, a variety of antibodyconjugates and methods are described in Trail et al., 1999, Curr. Opin.Immunol. 11:584-588, expressly incorporated by reference. Possibleconjugate partners include but are not limited to cytokines, cytotoxicagents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenicagents, a tyrosine kinase inhibitors. and other therapeutically activeagents. In some embodiments, conjugate partners may be thought of moreas payloads, that is to say that the goal of a conjugate is targeteddelivery of the conjugate partner to a targeted cell, for example acancer cell or immune cell, by the Ep-CAM targeting protein. Thus, forexample, the conjugation of a toxin to an anti-Ep-CAM antibody or Fcfusion targets the delivery of the toxin to cells expressing the Ep-CAMantigen. As will be appreciated by one skilled in the art, in realitythe concepts and defintions of fusion and conjugate are overlapping. Thedesignation of an Ep-CAM targeting protein as a fusion or conjugate isnot meant to constrain it to any particular embodiment of the presentinvention. Rather, these terms are used loosely to convey the broadconcept that any Ep-CAM targeting protein of the present invention maybe linked genetically, chemically, or otherwise, to one or morepolypeptides or molecules to provide some desireable property.

In one embodiment, the Ep-CAM targeting proteins of the presentinvention are fused or conjugated to a cytokine. By “cytokine” as usedherein is meant a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Forexample, as described in Penichet et al., 2001, J. Immunol. Methods248:91-101, expressly incorporated by reference, cytokines may be fusedto antibody to provide an array of desireable properties. Examples ofsuch cytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4,IL-5, IL-6, ILL7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; C5a; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

In an alternate embodiment, the EpCAM targeting proteins of the presentinvention are fused, conjugated, or operably linked to a toxin,including but not limited to small molecule toxins and enzymaticallyactive toxins of bacterial, fungal, plant or animal origin, includingfragments and/or variants thereof. For example, a variety ofimmunotoxins and immunotoxin methods are described in Thrush et al.,1996, Ann. Rev. Immunol. 14:49-71, expressly incorporated by reference.Small molecule toxins include but are not limited to calicheamicin,maytansine (U.S. Pat. No. 5,208,020, expressly incorporated byreference), trichothene, and CC1065. In one embodiment of the invention,the anti-Ep-CAM antibody or Fc fusion is conjugated to one or moremaytansine molecules (e.g. about 1 to about 10 maytansine molecules perantibody molecule). Maytansine may, for example, be converted toMay-SS-Me which may be reduced to May-SH3 and reacted with modifiedantibody or Fc fusion (Chari et al., 1992, Cancer Research 52: 127-131,expressly incorporated by reference) to generate a maytansinoid-antibodyor maytansinoid-Fc fusion conjugate. Another conjugate of interestcomprises an anti-Ep-CAM antibody or Fc fusion conjugated to one or morecalicheamicin molecules. The calicheamicin family of antibiotics arecapable of producing double-stranded DNA breaks at sub-picomolarconcentrations. Structural analogues of calicheamicin that may be usedinclude but are not limited to γ₁ ¹, α₂ ₁, α₃, N-acetyl-γ₁ ¹, PSAG, andΘ¹ ₁, (Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,1998, Cancer Research 58:2925-2928; U.S. Pat. Nos. 5,714,586; 5,712,374;5,264,586; 5,773,001; all expressly incorporated by reference).Dolastatin 10 analogs such as auristatin E (AE) and monomethylauristatinE (MMAE) may find use as conjugates for the Ep-CAM targeting proteins ofthe present invention (Doronina et al., 2003, Nat Biotechnol21(7):778-84; Francisco et al., 2003 Blood 102(4):1458-65; expresslyincorporated by reference). Useful enyzmatically active toxins includebut are not limited to diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordi proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin and the tricothecenes. See, for example, PCT WO93/21232, expressly incorporated by reference. The present inventionfurther contemplates a conjugate between an Ep-CAM targeting protein ofthe present invention and a compound with nucleolytic activity, forexample a ribonuclease or DNA endonuclease such as a deoxyribonuclease(Dnase).

In an alternate embodiment, an Ep-CAM targeting protein of the presentinvention may be fused, conjugated, or operably linked to a radioisotopeto form a radioconjugate. A variety of radioactive isotopes areavailable for the production of radioconjugate antibodies and Fcfusions. Examples include, but are not limited to, At211, I131, I125,Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu.See for example, reference.

In yet another embodiment, an targeti Ep-CAM ng protein of the presentinvention may be conjugated to a “receptor” (such streptavidin) forutilization in tumor pretargeting wherein the Ep-CAM targetingprotein-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide). In an alternateembodiment the Ep-CAM targeting protein is conjugated or operably linkedto an enzyme in order to employ Antibody Dependent Enzyme MediatedProdrug Therapy (ADEPT). ADEPT may be used by conjugating or operablylinking the Ep-CAM targeting protein to a prodrug-activating enzyme thatconverts a prodrug (e.g. a peptidyl chemotherapeutic agent, see PCT WO81/01145) to an active anti-cancer drug. See, for example, PCT WO88/07378 and U.S. Pat. No. 4,975,278; both expressly incorporated byreference. The enzyme component of the immunoconjugate useful for ADEPTincludes any enzyme capable of acting on a prodrug in such a way so asto covert it into its more active, cytotoxic form. Enzymes that areuseful in the method of this invention include but are not limited toalkaline phosphatase useful for converting phosphate-containing prodrugsinto free drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, useful for converting prodrugs that containD-amino acid substituents; carbohydrate-cleaving enzymes such as.beta.-galactosidase and neuramimidase useful for convertingglycosylated prodrugs into free drugs; beta-lactamase useful forconverting drugs derivatized with .alpha.-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes”, can be used to convert the prodrugs ofthe invention into free active drugs (see, for example, Massey, 1987,Nature 328: 457-458, expressly incorporated by reference). Ep-CAMtargeting protein-abzyme conjugates can be prepared for delivery of theabzyme to a tumor cell population. A variety of additional conjugatesare contemplated for the Ep-CAM targeting proteins of the presentinvention. A variety of chemotherapeutic agents, anti-angiogenic agents,tyrosine kinase inhibitors, and other therapeutic agents are describedbelow, which may find use as Ep-CAM targeting protein conjugates.

Also contemplated as fusion and conjugate partners are Fc polypeptides.Thus an Ep-CAM targeting protein may be a multimeric Fc polypeptide,comprising two or more Fc regions. The advantage of such a molecule isthat it provides multiple binding sites for Fc receptors with a singleprotein molecule. In one embodiment, Fc regions may be linked using achemical engineering approach. For example, Fab's and Fc's may be linkedby thioether bonds originating at cysteine residues in the hinges,generating molecules such as FabFc₂ (Kan et al., 2001, J. Immunol, 2001,166: 1320-1326; Stevenson et a., 2002, Recent Results Cancer Res. 159:104-12; U.S. Pat. No. 5,681,566; expressly incorporated by reference).Fc regions may be linked using disulfide engineering and/or chemicalcross-linking, for example as described in Caron et al., 1992, J. Exp,Med. 176:1191-1195, and Shopes, 1992, J. Immunol. 148(9):2918-22; bothexpressly incorporated by reference. In a preferred embodiment, Fcregions may be linked genetically, For example multiple Cγ2 domains havebeen fused between the Fab and Fc regions of an antibody (White et al.,2001, Protein Expression and Purification 21: 446-455, expresslyincorporated by reference). In a preferred embodiment, Fc regions in anEp-CAM targeting protein are linked genetically to generated tandemlylinked Fc regions as described in U.S. Ser. No. 60/531,752, filed Dec.22, 2003, entitled “Fc polypeptides with novel Fc receptor bindingsites”, expressly incorporated by reference. Tandemly linked Fcpolypeptides may comprise two or more Fc regions, preferably one tothree, most preferably two Fc regions. It may be advantageous to explorea number of engineering constructs in order to obtain homo- or hetero-tandemly linked Ep-CAM targeting proteins with the most favorablestructural and functional properties. Tandemly linked Ep-CAM targetingproteins may be homo- tandemly linked Ep-CAM targeting proteins, that isan Ep-CAM targeting protein of one isotype is fused genetically toanother Ep-CAM targeting protein of the same isotype. It is anticipatedthat because there are multiple FcγR, C1q, and/or FcRn binding sites ontandemly linked Fc polypeptides, effector functions and/orpharmacokinetics may be enhanced. In an alternate embodiment, Ep-CAMtargeting proteins from different isotypes may be tandemly linked,referred to as hetero-tandemly linked Ep-CAM targeting proteins. Forexample, because of the capacity to target FcγR and FcαRI receptors, anEp-CAM targeting protein that binds both FcγRs and FcαRI may provide asignificant clinical improvement.

Fusion and conjugate partners may be linked to any region of an Ep-CAMtargeting protein of the present invention, including at the N- orC-termini, or at some residue in-between the termini. In a preferredembodiment, a fusion or conjugate partner is linked at the N- orC-terminus of the Ep-CAM targeting protein, most preferably theNterminus. A variety of linkers may find use in the present invention tocovalently link Ep-CAM targeting proteins to a fusion or conjuatepartner or generate an Fc fusion. By “linker”, “linker sequence”,“spacer”, “tethering sequence” or grammatical equivalents thereof,herein is meant a molecule or group of molecules (such as a monomer orpolymer) that connects two molecules and often serves to place the twomolecules in a preferred configuration. A number of strategies may beused to covatently link molecules together. These include, but are notlimited to polypeptide linkages between N- and C-termini of proteins orprotein domains, linkage via disulfide bonds, and linkage via chemicalcross-linking reagents. In one aspect of this embodiment, the linker isa peptide bond, generated by recombinant techniques or peptidesynthesis. Choosing a suitable linker for a specific case where twopolypeptide chains are to be connected depends on various parameters,including but not limited to the nature of the two polypeptide chains(e.g., whether they naturally oligomerize), the distance between the N-and the C-termini to be connected if known, and/or the stability of thelinker towards proteolysis and oxidation. Furthermore, the linker maycontain amino acid residues that provide flexibility. Thus, the linkerpeptide may predominantly include the following amino acid residues:Gly, Ser, Ala, or Thr. The linker peptide should have a length that isadequate to link two molecules in such a way that they assume thecorrect conformation relative to one another so that they retain thedesired activity. Suitable lengths for this purpose include at least oneand not more than 50 amino acid residues Preferably, the linker is fromabout 1 to 30 amino acids in length, with linkers of 1 to 20 amino acidsin length being most preferred. In addition, the amino acid residuesselected for inclusion in the linker peptide should exhibit propertiesthat do not interfere significantly with the activity of thepolypeptide. Thus, the linker peptide on the whole should not exhibit acharge that would be inconsistent with the activity of the polypeptide,or interfere with internal folding, or form bonds or other interactionswith amino acid residues in one or more of the monomers that wouldseriously impede the binding of receptor monomer domains. Useful linkersinclude glycine-serine polymers (including, for example, (GS)n, (GSGGS)n(SEQ ID NO:168), (GGGGS)n (SEQ ID NO:169), and (GGGS)n (SEQ ID NO:170),where n is an integer of at least one), glycine-alanine polymers,alanine-serine polymers, and other flexible linkers such as the tetherfor the shaker potassium channel, and a large variety of other flexiblelinkers, as will be appreciated by those in the art. Glycine-serinepolymers are preferred since both of these amino acids are relativelyunstructured, and therefore may be able to serve as a neutral tetherbetween components. Secondly, serine is hydrophilic and therefore ableto solubilize what could be a globular glycine chain. Third, similarchains have been shown to be effective in joining subunits ofrecombinant proteins such as single chain antibodies. Suitable Linkersmay also be identified by screening databases of known three-dimensionalstructures for naturally occurring motifs that can bridge the gapbetween two polypeptide chains. In a preferred embodiment, the linker isnot immunogenic when administered in a human patient. Thus linkers maybe chosen such that they have low immunogenicity or are thought to havelow immunogenicity. For example, a linker may be chosen that existsnaturally in a human. In a most preferred embodiment, the linker has thesequence of the hinge region of an antibody, that is the sequence thatlinks the antibody Fab and Fc regions; alternatively the linker has asequence that comprises part of the hinge region, or a sequence that issubstantially similar to the hinge region of an antibody. Another way ofobtaining a suitable linker is by optimizing a simple linker, e.g.,(Gly4Ser)n, through random mutagenesis. Alternatively, once a suitablepolypeptide linker is defined, additional linker polypeptides can becreated to select amino acids that more optimally interact with thedomains being linked. Other types of linkers that may be used in thepresent invention include artificial polypeptide linkers and inteins. Inanother embodiment, disulfide bonds are designed to link the twomolecules. In another embodiment, linkers are chemical cross-linkingagents. For example, a variety of bifunctional protein coupling agentsmay be used, including but not limited toN-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., 1971, Science 238:1098,expressly incorporated by reference. Chemical linkers may enablechelation of an isotope. For example, Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucteotide to the antibody (see PCT WO 94/11026). The linker may becleavable, facilitating release of the cytotoxic drug in the cell. Forexample, an acid-labile linker, peptidase-sensitive linker, dimethyllinker or disulfide-containing linker (Chari et al., 1992, CancerResearch 52: 127-131, expressly incorporated by reference) may be used.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers, that is may find use to link the Ep-CAMtargeting proteins of the present invention to a fusion or conjugatepartner to generate an anti-Ep-CAM Fc fusion, or to link the Ep-CAMtargeting proteins of the present invention to a conjugate.

Experimental Production of Ep-CAM Targeting Proteins

The present invention provides methods for producing and experimentallytesting Ep-CAM targeting proteins. The described methods are not meantto constrain the present invention to any particular application ortheory of operation. Rather, the provided methods are meant toillustrate generally that one or more Ep-CAM targeting proteins may beproduced and experimentally tested to obtain variant Ep-CAM targetingproteins. General methods for antibody molecular biology, expression,purification, and screening are described in Antibody Engineering,edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001; andHayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; Antibodies: A LaboratoryManual by Harlow & Lane, New York: Cold Spring Harbor Laboratory Press,1988; all expressly incorporated by reference.

In one embodiment of the present invention, nucleic acids are createdthat encode the Ep-CAM targeting proteins, and that may then be clonedinto host cells, expressed and assayed, if desired. Thus, nucleic acids,and particularly DNA, may be made that encode each protein sequence.These practices are carried out using well-known procedures. Forexample, a variety of methods that may find use in the present inventionare described in Molecular Cloning—A Laboratory Manual, 3_(rd) Ed.(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), andCurrent Protocols in Molecular Biology (John Wiley & Sons); bothexpressly incorporated by reference. As will be appreciated by thoseskilled in the art, the generation of exact sequences for a librarycomprising a large number of sequences is potentially expensive and timeconsuming. Accordingly, there are a variety of techniques that may beused to efficiently generate libraries of the present invention. Suchmethods that may find use in the present invention are described orreferenced in U.S. Pat. No. 6,403,312; U.S. Ser. Nos. 09/782,004;09/927,790; 10/218,102; PCT WO 01/40091; and PCT WO 02/25588; allexpressly incorporated by reference. Such methods include but are notlimited to gene assembly methods, PCR-based method and methods which usevariations of PCR, ligase chain reaction-based methods, pooled oligomethods such as those used in synthetic shuffling, error-proneamplification methods and methods which use oligos with randommutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in the present invention forgenerating nucleic acids that encode Ep-CAM targeting proteins.

The Ep-CAM targeting proteins of the present invention may be producedby culturing a host cell transformed with nucleic acid, preferably anexpression vector, containing nucleic acid encoding the Ep-CAM targetingproteins, under the appropriate conditions to induce or cause expressionof the protein. The conditions appropriate for expression will vary withthe choice of the expression vector and the host cell, and will beeasily ascertained by one skilled in the art through routineexperimentation. A wide variety of appropriate host cells may be used,including but not limited to mammalian cells, bacteria, insect cells,and yeast. For example, a variety of cell lines that may find use in thepresent invention are described in the ATCC® cell line catalog,available from the American Type Culture Collection.

in a preferred embodiment, the Ep-CAM targeting proteins are expressedin mammalian expression systems, including systems in which theexpression constructs are introduced into the mammalian cells usingvirus such as retrovirus or adenovirus. Any mammalian cells may be used,with human, mouse, rat, hamster, and primate cells being particularlypreferred. Suitable cells also include known research cells, includingbut not limited to Jurkat T cells, NIH3T3, CHO, BHK, COS, HEK293, PERC.6, HeLa, Sp2/0, NS0 cells and variants thereof. In an alternatelypreferred embodiment, library proteins are expressed in bacterial cells.Bacterial expression systems are well known in the art, and includeEscherichia coli (E. coli), Bacillus subtilis, Streptococcus cremoris,and Streptococcus lividans. In alternate embodiments, Ep-CAM targetingproteins are produced in insect cells (e.g. sf21/Sf9, Trichoplusia niBti-Tn5b1-4) or yeast cells (e.g. S. cerevsiae, Picha, etc). In analternate embodiment, Ep-CAM targeting proteins are expressed in vitrousing cell free translation systems. In vitro translation systemsderived from both prokaryotic (e.g. E. coli) and eukaryotic (e.g. wheatgerm, rabbit reticulocytes) cells are available and may be chosen basedon the expression levels and functional properties of the protein ofinterest. For example, as appreciated by those skilled in the art, invitro translation is required for some display technologies, for exampleribosome display. In addition, the Ep-CAM targeting proteins may beproduced by chemical synthesis methods. Also transgenic expressionsystems both animal (e.g. cow, sheep or goat milk, embryonated hen'seggs, whole insect larvae, etc.) and plant (e.g. corn, tobacco,duckweed, etc.)

The nucleic acids that encode the Ep-CAM targeting proteins of thepresent invention may be incorporated into an expression vector in orderto express the protein. A variety of expression vectors may be utilizedfor protein expression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in the present inventioninclude but are not limited to those which enable protein expression inmammalian cells, bacteria insect cells, yeast, and in in vitro systems.As is known in the art, a variety of expression vectors are available,commercially or otherwise, that may find use in the present inventionfor expressing Ep-CAM targeting proteins.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the Ep-CAM targetingprotein, and are typically appropriate to the host cell used to expressthe protein. In general, the transcriptional and translationalregulatory sequences may include promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. As is also known inthe art, expression vectors typically contain a selection gene or markerto allow the selection of transformed host cells containing theexpression vector. Selection genes are well known in the art and willvary with the host cell used.

Ep-CAM targeting proteins may be operably linked to a fusion partner toenable targeting of the expressed protein, purification, screening,display, and the like. Fusion partners may be linked to the Ep-CAMtargeting protein sequence via a linker sequences. The linker sequencewill generally comprise a small number of amino acids, typically lessthan ten, although longer linkers may also be used. Typically, linkersequences are selected to be flexible and resistant to degradation. Aswill be appreciated by those skilled in the art, any of a wide varietyof sequences may be used as linkers. For example, a common linkersequence comprises the amino acid sequence GGGGS (SEQ ID NO:169). Afusion partner may be a targeting or signal sequence that directs Ep-CAMtargeting protein and any associated fusion partners to a desiredcellular location or to the extracellular media. As is known in the art,certain signaling sequences may target a protein to be either secretedinto the growth media, or into the periplasmic space, located betweenthe inner and outer membrane of the cell. A fusion partner may also be asequence that encodes a peptide or protein that enables purificationand/or screening. Such fusion partners include but are not limited topolyhistidine tags (His-tags) (for example H₆ and H₁₀ or other tags foruse with Immobilized Metal Affinity Chromatography (IMAC) systems (e.g.Ni⁺² affinity columns)), GST fusions, MBP fusions, Strep-tag, the BSPbiotinylation target sequence of the bacterial enzyme BirA, and epitopetags which are targeted by antibodies (for example c-myc tags,flag-tags, and the like). As will be appreciated by those skilled in theart, such tags may be useful for purification, for screening, or both.For example, an Ep-CAM targeting protein may be purified using a His-tagby immobilizing it to a Ni⁺² affinity column, and then afterpurification the same His-tag may be used to immobilize the antibody toa Ni⁺² coated plate to perform an ELISA or other binding assay (asdescribed below). A fusion partner may enable the use of a selectionmethod to screen Ep-CAM targeting proteins (see below). Fusion partnersthat enable a variety of selection methods are well-known in the art,and all of these find use in the present invention. For example, byfusing the members of an Ep-CAM targeting protein library to the geneIII protein, phage display can be employed (Kay et at. Phage display ofpeptides and proteins: a laboratory manual, Academic Press, San Diego,Calif., 1996; Lowman et al., 1991, Biochemistry 30:10832-10838; Smith,1985, Science 228:1315-1317; all expressly incorporated by reference).Fusion partners may enable Ep-CAM targeting proteins to be labeled.Alternatively, a fusion partner may bind to a specific sequence on theexpression vector, enabling the fusion partner and associated Ep-CAMtargeting protein to be linked covalently or noncovalently with thenucleic acid that encodes them. For example, U.S. Ser. Nos. 09/642,574;10/080,376; 09/792,630; 10/023,208; 09/792,626; 10/082,671; 09/953,351;10/097,100; 60/366,658; PCT WO 00/22906; PCT WO 01/49058; PCT WO02/04852; PCT WO 02/04853; PCT WO 02/08023; PCT WO 01/28702; and PCT WO02/07466, all expressly incorporated by reference, describe such afusion partner and technique that may find use in the present invention.

The methods of introducing exogenous nucleic acid into host cells arewell known in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In a preferred embodiment, Ep-CAM targeting proteins are purified orisolated after expression. Proteins may be isolated or purified in avariety of ways known to those skilled in the art. Standard purificationmethods include chromatographic techniques, including ion exchange,hydrophobic interaction, affinity, sizing or gel filtration, andreversed-phase, carried out at atmospheric pressure or at high pressureusing systems such as FPLC and HPLC. Purification methods also includeelectrophoretic, immunological, precipitation, dialysis, andchromatofocusing techniques. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.As is well known in the art, a variety of natural proteins bind Fc andantibodies, and these proteins can find use in the present invention forpurification of Ep-CAM targeting proteins. For example, the bacterialproteins A and G bind to the Fc region. Likewise, the bacterial proteinL binds to the Fab region of some antibodies, as of course does theantibody's target antigen. Purification can often be enabled by aparticular fusion partner. For example, Ep-CAM targeting proteins may bepurified using glutathione resin if a GST fusion is employed, Ni⁺²affinity chromatography if a His-tag is employed, or immobilizedanti-flag antibody if a flag-tag is used. For general guidance insuitable purification techniques, see Protein Purification: Principlesand Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994. The degree ofpurification necessary will vary depending on the screen or use of theEp-CAM targeting proteins. In some instances no purification isnecessary. For example in one embodiment, if the Ep-CAM targetingproteins are secreted, screening may take place directly from the media.As is well known in the art, some methods of selection do not involvepurification of proteins. Thus, for example, if a library of Ep-CAMtargeting proteins is made into a phage display library, proteinpurification may not be performed.

Experimental Testing of Ep-CAM Targeting Proteins

Assays

Ep-CAM targeting proteins may be screened using a variety of methods,including but not limited to those that use in vitro assays, in vivo andcell-based assays, and selection technologies. Automation andhigh-throughput screening technologies may be utilized in the screeningprocedures. Screening may employ the use of a fusion partner or label.The use of fusion partners has been discussed above. By “labeled” hereinis meant that the Ep-CAM targeting proteins of the invention have one ormore elements, isotopes, or chemical compounds attached to enable thedetection in a screen. In general, labels fall into three classes: a)immune labels, which may be an epitope incorporated as a fusion partnerthat is recognized by an antibody, b) isotopic labels, which may beradioactive or heavy isotopes, and c) small molecule labels, which mayinclude fluorescent and colorimetric dyes, or molecules such as biotinthat enable other labeling methods. Labels may be incorporated into thecompound at any position and may be incorporated in vitro or in vivoduring protein expression.

In a preferred embodiment, the functional and/or biophysical propertiesof Ep-CAM targeting proteins are screened in an in vitro assay. In vitroassays may allow a broad dynamic range for screening properties ofinterest. Properties of Ep-CAM targeting proteins that may be screenedinclude but are not limited to stability, solubility, and affinity forFc ligands, for example FcγRs. Multiple properties may be screenedsimultaneously or individually. Proteins may be purified or unpurified,depending on the requirements of the assay. In one embodiment, thescreen is a qualitative or quantitative binding assay for binding ofEp-CAM targeting proteins to a protein or nonprotein molecule that isknown or thought to bind the Ep-CAM targeting protein. In a preferredembodiment, the screen is a binding assay for measuring binding to theEp-CAM target antigen. In an alternately preferred embodiment, thescreen is an assay for binding of Ep-CAM targeting proteins to an Fcligand, including but are not limited to the family of FcγRs, theneonatal receptor FcRn, the complement protein C1q, and the bacterialproteins A and G. The Fc ligands may be from any organism, with humans,mice, rats, rabbits, and monkeys preferred. Binding assays can becarried out using a variety of methods known in the art, including butnot limited to FRET (Fluorescence Resonance Energy Transfer) and BRET(Bioluminescence Resonance Energy Transfer)-based assays, AlphaScreen™(Amplified Luminescent Proximity Homogeneous Assay), ScintillationProximity Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (SurfacePlasmon Resonance, also known as BIACORE®), isothermal titrationcalorimetry, differential scanning calorimetry, gel electrophoresis, andchromatography including gel filtration. These and other methods maytake advantage of some fusion partner or label of the Ep-CAM targetingprotein. Assays may employ a variety of detection methods including butnot limited to chromogenic, fluorescent, luminescent, or isotopiclabels.

The biophysical properties of Ep-CAM targeting proteins, for examplestability and solubility, may be screened using a variety of methodsknown in the art. Protein stability may be determined by measuring thethermodynamic equilibrium between folded and unfolded states. Forexample, Ep-CAM targeting proteins of the present invention may beunfolded using chemical denaturant, heat, or pH, and this transition maybe monitored using methods including but not limited to circulardichroism spectroscopy, fluorescence spectroscopy, absorbancespectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As will beappreciated by those skilled in the art, the kinetic parameters of thefolding and unfolding transitions may also be monitored using these andother techniques. The solubility and overall structural integrity of anEp-CAM targeting protein may be quantitatively or qualitativelydetermined using a wide range of methods that are known in the art,Methods which may find use in the present invention for characterizingthe biophysical properties of Ep-CAM targeting proteins include gelelectrophoresis, isoelectric focusing, capillary electrophoresis,chromatography such as size exclusion chromatography, ion-exchangechromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an Ep-CAM targeting protein could beassayed for its ability to bind target antigen, then exposed to elevatedtemperature for one or more defined periods of time, then assayed forantigen binding again. Because unfolded and aggregated protein is notexpected to be capable of binding antigen, the amount of activityremaining provides a measure of the Ep-CAM targeting protein's stabilityand solubility.

In a preferred embodiment, the library is screened using one or morecell-based or in vitro assays. For such assays, Ep-CAM targetingproteins, purified or unpurified, are typically added exogenously suchthat cells are exposed to individual variants or groups of variantsbelonging to a library. These assays are typically, but not always,based on the biology of the ability of the anti-Ep-CAM antibody or Fcfusion to bind to Ep-CAM and mediate some biochemical event, for exampleeffector functions like cellular lysis, phagocytosis, ligand/receptorbinding inhibition, inhibition of growth and/or proliferation, and thelike. Such assays often involve monitoring the response of cells toEp-CAM targeting protein, for example cell survival, cell death,cellular phagocytosis, cell lysis, change in cellular morphology, ortranscriptional activation such as cellular expression of a natural geneor reporter gene. For example, such assays may measure the ability ofEp-CAM targeting proteins to elicit ADCC, ADCP, or CDC. For some assaysadditional cells or components, that is in addition to the target cells,may need to be added, for example example serum complement, or effectorcells such as peripheral blood monocytes (PBMCs), NK cells, macrophages,and the like. Such additional cells may be from any organism, preferablyhumans, mice, rat, rabbit, and monkey. Crosslinked or monomericantibodies and Fc fusions may cause apoptosis of certain cell linesexpressing the antibody's target antigen, or they may mediate attack ontarget cells by immune cells which have been added to the assay. Methodsfor monitoring cell death or viability are known in the art, and includethe use of dyes, fluorophores, immunochemical, cytochemical, andradioactive reagents. For example, caspase assays orannexin-flourconjugates may enable apoptosis to be measured, and uptakeor release of radioactive substrates (e.g. Chromium-51 release assays)or the metabolic reduction of fluorescent dyes such as alamar blue mayenable cell growth, proliferationor activation to be monitored. In apreferred embodiment, the DELFIA® EuTDA-based cytotoxicity assay (PerkinElmer, MA.) is used. Alternatively, dead or damaged target cells may bemonitoried by measuring the release of one or more natural intracellularproteins, for example lactate dehydrogenase. Transcriptional activationmay also serve as a method for assaying function in cell-based assays.In this case, response may be monitored by assaying for natural genes orproteins which may be upregulated or down-regulated, for example therelease of certain interleukins may be measured, or alternativelyreadout may be via a luciferase or GFP-reporter construct. Cell-basedassays may also involve the measure of morphological changes of cells asa response to the presence of an EP-CAM targeting protein. Cell typesfor such assays may be prokaryotic or eukaryotic, and a variety of celllines that are known in the art may be employed. Alternatively,cell-based screens are performed using cells that have been transformedor transfected with nucleic acids encoding the Ep-CAM targetingproteins.

In vitro assays include but are not limited to binding assays, ADCC,CDC, cytotoxicity, proliferation, peroxide/ozone release, chemotaxis ofeffector cells, inhibition of such assays by reduced effector functionantibodies; ranges of activities such as >100× improvement or >100×reduction, blends of receptor activation and the assay outcomes that areexpected from such receptor profiles.

Animal Models

The biological properties of the Ep-CAM targeting proteins of thepresent invention may be characterized in cell, tissue, and wholeorganism experiments. As is know in the art, drugs are often tested inanimals, including but not limited to mice, rats, rabbits, dogs, cats,pigs, and monkeys, in order to measure a drug's efficacy for treatmentagainst a disease or disease model, or to measure a drug'spharmacokinetics, toxicity, and other properties. The animals may bereferred to as disease models. With respect to the Ep-CAM targetingproteins of the present invention, a particular challenge arises whenusing animal models to evaluate the potential for in-human efficacy ofcandidate polypeptides—this is due, at least in part, to the fact thatEp-CAM targeting proteins that have a specific effect on the affinityfor a human Fc receptor may not have a similar affinity effect with theorthologous animal receptor. These problems can be further exacerbatedby the inevitable ambiguities associated with correct assignment of trueorthologues (Mechetina et al., Immunogenetics, 2002 54:463-468,expressly incorporated by reference), and the fact that some orthologuessimply do not exist in the animal (e g. humans possess an FcRIIa whereasmice do not). Therapeutics are often tested in mice, including but notlimited to nude mice, SCID mice, xenograft mice, and transgenic mice(including knockins and knockouts). For example, an anti-Ep-CAM antibodyor Fc fusion of the present invention that is intended as an anti-cancertherapeutic may be tested in a mouse cancer model, for example axenograft mouse. In this method, a tumor or tumor cell line is graftedonto or injected into a mouse, and subsequently the mouse is treatedwith the therapeutic to determine the ability of the anti-Ep-CAMantibody or Fc fusion to reduce or inhibit cancer growth and metastasis.An alternative approach is the use of a SCID murine model in whichimmune-deficient mice are injected with human PBLs, conferring asemi-functional and human immune—system with an appropriate array ofhuman FcRs—to the mice that have subsequently been injected withantibodies or Fc-polypeptides that target injected human tumor cells. Insuch a model, the Fc-polypeptides that target the desired antigen (suchas her2/neu on SkOV3 ovarian cancer cells) interact with human PBLswithin the mice to engage tumoricidal effector functions. Suchexperimentation may provide meaningful data for determination of thepotential of the Ep-CAM targeting protein to be used as a therapeutic,Any organism, preferably mammals, may be used for testing. For examplebecause of their genetic similarity to humans, monkeys can be suitabletherapeutic models, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the anti-Ep-CAM antibodies and Fcfusions of the present invention. Tests of the Ep-CAM targeting proteinsof the present invention in humans are ultimately required for approvalas drugs, and thus of course these experiments are contemplated. Thusthe Ep-CAM targeting proteins of the present invention may be tested inhumans to determine their therapeutic efficacy, toxicity,pharmacokinetics, and/or other clinical properties.

The Ep-CAM targeting proteins of the present invention may confersuperior performance on Fc-containing therapeutics in animal models orin humans. The receptor binding profiles of such Ep-CAM targetingproteins, as described in this specification, may, for example, beselected to increase the potency of cytotoxic drugs or to targetspecific effector functions or effector cells to improve the selectivityof the drug's action. Further, receptor binding profiles can be selectedthat may reduce some or all effector functions thereby reducing theside-effects or toxicity of such Fc-containing drug, For example, anEp-CAM targeting protein with reduced binding to FcγRIIIa, FcγRI andFcγRIIa can be selected to eliminate most cell-mediated effectorfunction, or an Ep-CAM targeting protein with reduced binding to C1q maybe selected to limit complement-mediated effector functions. In somecontexts, such effector functions are known to have potential toxiceffects, therefore eliminating them may increase the safety of theFc-bearing drug and such improved safety may be characterized in animalmodels. In some contexts, such effector functions are known to mediatethe desirable therapeutic activity, therefore enhancing them mayincrease the activity or potency of the Fc-bearing drug and suchimproved activity or potency may be characterized in animal models.

Optimized Ep-CAM targeting proteins can be tested in a variety oforthotopic tumor models. These clinically relevant animal models areimportant in the study of pathophysiology and therapy of aggressivecancers like pancreatic, prostate and breast cancer. Immune deprivedmice including, but not limited to athymic nude or SCID mice arefrequently used in scoring of local and systemic tumor spread from thesite of intraorgan (e.g. pancreas, prostate or mammary gland) injectionof human tumor cells or fragments of donor patients.

In preferred embodiments, EP-CAM targeting proteins of the presentinvention may be assessed for efficacy in clinically relevant animalmodels of various human diseases. In many cases, relevant models includevarious transgenic animals for specific tumor antigens

Relevant transgenic models such as those that express human Fc receptors(e.g., CD16 including the gamma chain, FCγR1, RIIa/b, and others) couldbe used to evaluate and test Ep-CAM targeting protein antibodies andFc-fusions in their efficacy. The evalution of Ep-CAM targeting proteinsby the introduction of human genes that directly or indirectly mediateeffector function in mice or other rodents that may enable physiologicalstudies of efficacy in tumor toxicity or other diseases such asautoimmune disorders and RA. Human Fc receptors such as FCγRIIIa maypossess polymorphisms such as that in position 158 V or F which wouldfurther enable the introduction of specific and combinations of humanpolymorphisms into rodents. The various studies involvingpolymorphism-specific FcRs is not limited to this section, howeverencompasses all discussions and applications of FcRs in general asspecficied in throughout this application. Ep-CAM targeting proteins ofthe present invention may confer superior activity on Fc-containingdrugs in such transgenic models, in particular variants with bindingprofiles optimized for human FcγRIIIa mediated activity may showsuperior activity in transgenic CD16 mice. Similar improvements inefficacy in mice transgenic for the other human Fc receptors, e.g.FcγRIIa, FcγRI, etc., may be observed for Ep-CAM targeting proteins withbinding profiles optimized for the respective receptors. Mice transgenicfor multiple human receptors would show improved activity for Ep-CAMtargeting proteins with binding profiles optimized for the correspondingmultiple receptors, for example as outlined in Table 1.

Because of the difficulties and ambiguities associated with using animalmodels to characterize the potential efficacy of candidate therapeuticantibodies in a human patient, some variant polypeptides of the presentinvention may find utility as proxies for assessing potential in-humanefficacy. Such proxy molecules would preferably mimic—in the animalsystem—the FcR and/or complement biology of a corresponding candidatehuman Ep-CAM targeting protein. This mimicry is most likely to bemanifested by relative association affinities between specific Ep-CAMtargeting proteins and animal vs. human receptors. For example, if onewere using a mouse model to assess the potential in-human efficacy of anEp-CAM targeting protein that has enhanced affinity for human FcRIIIa,an appropriate proxy variant would have enhanced affinity for mouseFcRIII-2 (mouse CD16-2). Alternatively if one were using a mouse modelto assess the potential in-human efficacy of an Ep-CAM targeting proteinthat has reduced affinity for the inhibitory human FcRIIb, anappropriate proxy variant would have reduced affinity for mouse FcRII.It should also be noted that the proxy Ep-CAM targeting proteins couldbe created in the context of a human Ep-CAM targeting protein, an animalEp-CAM targeting protein, or both.

In a preferred embodiment, the testing of Ep-CAM targeting proteins mayinclude study of efficacy in primates (e.g. cynomolgus monkey model) tofacilitate the evaluation of depletion of specific target cellsharboring Ep-CAM antigen. Additional primate models include but notlimited to that of the rhesus monkey and Fc polypetides in therapeuticstudies of autoimmune, transplantation and cancer,

Toxicity studies are performed to determine the antibody or Fc-fusionrelated-effects that cannot be evaluated in standard pharmacologyprofile or occur only after repeated administration of the agent. Mosttoxicity tests are performed in two species—a rodent and a non-rodent—toensure that any unexpected adverse effects are not overlooked before newtherapeutic entities are introduced into man. In general, these modelsmay measure a variety of toxicities including genotoxicity, chronictoxicity, immunogenicity, reproductive/developmental toxicity andcarcinogenicity. Included within the aforementioned parameters arestandard measurement of food consumption, bodyweight, antibodyformation, clinical chemistry, and macro- and microscopic examination ofstandard organs/tissues (e.g. cardiotoxicity). Additional parameters ofmeasurement are injection site trauma and the measurement ofneutralizing antibodies, if any. Traditionally, monoclonal antibodytherapeutics, naked or conjugated are evaluated for cross-reactivitywith normal tissues, immunogenicity/antibody production, conjugate orlinker toxicity and “bystander” toxicity of radiolabeled species.Nonetheless, such studies may have to be individualized to addressspecific concerns and following the guidance set by ICH S6 (Safetystudies for biotechnological products also noted above). As such, thegeneral principles are that the products are sufficiently wellcharacterized and for which impuritiestcontaminants have been removed,that the test material is comparable throughout development, and GLPcompliance.

The pharmacokinetics (PK) of the Ep-CAM targeting proteins of theinvention can be studied in a variety of animal systems, with the mostrelevant being non-human primates such as the cynomolgus, rhesusmonkeys. Single or repeated i.v. or s.c. administrations over a doserange of 6000-fold (0.05-300 mg/kg) can be evaluated for the half-life(days to weeks) using plasma concentration and clearance as well asvolume of distribution at a steady state and level of systemicabsorbance can be measured. Examples of such parameters of measurementgenerally include maximum observed plasma concentration (Cmax), the timeto reach Cmax (Tmax), the area under the plasma concentration-time curvefrom time 0 to infinity [AUC(0-inf] and apparent elimination half-life(T1/2). Additional measured prameters could include compartmentalanalysis of concentration-time data obtained following i.v.administration and bioavailability. Examples ofpharmacological/toxicological studies using cynomolgus have beenestablished for Rituxan and Zevatin in which monoclonal antibodies toCD20 are cross-reactive. Biodistribution, dosimetry (for radiolabledantibodies or Fc fusions), and PK studies can also be done in rodentmodels. Such studies would evaluate tolerance at all doses administered,toxicity to local tissues, preferential localization to rodent xenograftanimal models, depletion of target cells (e.g. CD20 positive cells).

The Ep-CAM targeting proteins of the present invention may confersuperior pharmacokinetics on Fc-containing therapeutics in animalsystems or in humans. For example, increased binding to FcRn mayincrease the half-life and exposure of the Fc-containing drug.Alternatively, decreased binding to FcRn may decrease the half-life andexposure of the Fc-containing drug in cases where reduced exposure isfavorable such as when such drug has side-effects.

It is known in the art that the array of Fc receptors is differentiallyexpressed on various immune cell types, as well as in different tissues.Differential tissue distribution of Fc receptors may ultimately have animpact on the pharmacodynamic (PD) and pharmacokinetic (PK) propertiesof EP-CAM targeting proteins of the present invention. Because Ep-CAMtargeting proteins of the presentation have varying affinities for thearray of Fc receptors, further screening of the polypeptides for PDand/or PK properties may be extremely useful for defining the optimalbalance of PD, PK, and therapeutic efficacy conferred by each candidatepolypeptide.

Pharmacodynamic studies may include, but are not limited to, targetingspecific tumor cells or blocking signaling mechanisms, measuringdepletion of target antigen expressing cells or signals, etc. The Ep-CAMtargeting proteins of the present invention may target particulareffector cell populations and thereby direct Fc-containing drugs torecruit certain activities to improve potency or to increase penetrationinto a particularly favorable physiological compartment. For example,neutrophil activity and localization can be targeted by an Ep-CAMtargeting protein that preferentially targets FcγRIIIb. Suchpharmacodynamic effects may be demonstrated in animal models or inhumans.

Clinical Use of EP-CAM Targeting Proteins

The Ep-CAM targeting proteins of the present invention may be used forvarious therapeutic purposes. As will be appreciated by those skilled inthe art, the Ep-CAM targeting proteins of the present invention may beused for any therapeutic purpose that antibodies, Fc fusions. and thelike may be used for. In a preferred embodiment, the Ep-CAM targetingproteins are administered to a patient to treat disorders including butnot limited to cancer.

A “patient” for the purposes of the present invention includes bothhumans and other animals, preferably mammals and most preferably humans.Thus the Ep-CAM targeting proteins of the present invention have bothhuman therapy and veterinary applications. The term “treatment” in thepresent invention is meant to include therapeutic treatment, as well asprophylactic, or suppressive measures for a disease or disorder. Thus,for example, successful administration of an EP-CAM targeting proteinprior to onset of the disease results in treatment of the disease. Asanother example, successful administration of an optimized Ep-CAMtargeting protein after clinical manifestation of the disease to combatthe symptoms of the disease comprises treatment of the disease.“Treatment” also encompasses administration of an optimized Ep-CAMtargeting protein after the appearance of the disease in order toeradicate the disease. Successful administration of an agent after onsetand after clinical symptoms have developed, with possible abatement ofclinical symptoms and perhaps amelioration of the disease, comprisestreatment of the disease. Those “in need of treatment” include mammalsalready having the disease or disorder, as well as those prone to havingthe disease or disorder, including those in which the disease ordisorder is to be prevented.

Diseases

In one embodiment, an Ep-CAM targeting protein of the present inventionis administered to a patient having a disease involving inappropriateexpression of a protein or other molecule. Within the scope of thepresent invention this is meant to include diseases and disorderscharacterized by aberrant proteins, due for example to alterations inthe amount of a protein present, protein localization, posttranslationalmodification, conformational state, the presence of a mutant or pathogenprotein, etc. Similarly, the disease or disorder may be characterized byalterations molecules including but not limited to polysaccharides andgangliosides. An overabundance may be due to any cause, including butnot limited to overexpression at the molecular level, prolonged oraccumulated appearance at the site of action, or increased activity of aprotein relative to normal. Included within this definition are diseasesand disorders characterized by a reduction of a protein. This reductionmay be due to any cause, including but not limited to reduced expressionat the molecular level, shortened or reduced appearance at the site ofaction, mutant forms of a protein, or decreased activity of a proteinrelative to normal. Such an overabundance or reduction of a protein canbe measured relative to normal expression, appearance, or activity of aprotein, and the measurement may play an important role in thedevelopment and/or clinical testing of the Ep-CAM targeting proteins ofthe present invention.

By “cancer” and “cancerous” herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma),neuroendocrine tumors, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

More particular examples of such cancers include hematologicmalignancies, such as Hodgkin's lymphoma; non-Hodgkin's lymphomas(Burkitt's lymphoma, small lymphocytic lymphomalchronic lymphocyticleukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma,diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cellleukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursorcells, including B-cell acute lymphoblastic leukemiallymphoma, andT-cell acute lymphoblastic leukemiallymphoma, thymoma, tumors of themature T and NK cells, including peripheral T-cell leukemias, adultT-cell leukemia/T-cell lymphomas and large granular lymphocyticleukemia, Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia; tumors of the central nervous system such as glioma,glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma,and retinoblastoma; solid tumors of the head and neck (eg.nasopharyngeal cancer, salivary gland carcinoma, and esophagael cancer),lung (eg. small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung),digestive system (eg. gastric or stomach cancer includinggastrointestinal cancer, cancer of the bile duct or biliary tract, coloncancer, rectal cancer, colorectal cancer, and anal carcinoma),reproductive system (eq. testicular, penile, or prostate cancer,uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer),skin (eg. melanoma, basal cell carcinoma, squamous cell cancer, actinickeratosis), liver (eg. liver cancer, hepatic carcinoma, hepatocellularcancer, and hepatoma), bone (eq. osteoclastoma, and osteolytic bonecancers) additional tissues and organs (eg. pancreatic cancer, bladdercancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer ofthe peritoneum, and Kaposi's sarcoma), and tumors of the vascular system(eg. angiosarcoma and hemagiopericytoma).

By “autoimmune diseases” herein include allogenic islet graft rejection,alopecia areata, ankylosing spondylitis, antiphosphoiipid syndrome,autoimmune Addison's disease, antineutrophil cytoplasmic autoantibodies(ANCA), autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune myocarditis, autoimmuneneutropenia, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, autoimmune urticaria, Behcet's disease, bullouspemphigoid, cardiomyopathy, Castleman's syndrome, celiacspruce-dermatitis, chronic fatigue immune disfunction syndrome, chronicinflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn'sdisease, dermatomyositis, discoid lupus, essential mixedcryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis,glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture'ssyndrome, graft-versus-host disease (GVHD), Hashiimoto's thyroiditis,hemophilia A, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, IgM polyneuropathies, immune mediatedthrombocytopenia, juvenile arthritis, Kawasaki's disease, lichenplantus, lupus erthematosis, Meniere's disease, mixed connective tissuedisease, multiple sclerosis, type 1 diabetes mellitus, myastheniagravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychrondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobinulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Reynauld'sphenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis,scleroderma, Sjorgen's syndrome, solid organ transplant rejection,stiff-man syndrome, systemic lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, thrombotic thrombocytopeniapurpura, ulcerative colitis, uveitis, vasculitides such as dermatitisherpetiformis vasculitis, vitiligo, and Wegner's granulomatosis.

By “inflammatory disorders” herein include acute respiratory distresssyndrome (ARDS), acute septic arthritis, allergic encephalomyelitis,allergic rhinitis, allergic vasculitis, allergy, asthma,atherosclerosis, chronic inflammation due to chronic bacterial or viralinfectionis, chronic obstructive pulmonary disease (COPD), coronaryartery disease, encephalitis, inflammatory bowel disease, inflammatoryosteolysis, inflammation associated with acute and delayedhypersensitivity reactions, inflammation associated with tumors,peripherali nerve injury or demyelinating diseases, inflammationassociated with tissue trauma such as burns and ischemia, inflammationdue to meningitis, multiple organ injury syndrome, pulmonary fibrosis,sepsis and septic shock, Stevens-Johnson syndrome, undifferentiatedarthropy, and undifferentiated spondyloarthropathy.

By “infectious diseases” herein include diseases caused by pathogenssuch as viruses, bacteria, fungi, protozoa, and parasites. Infectiousdiseases may be caused by viruses including adenovirus, cytomegalovirus,dengue, Epstein-Barr, hanta, hepatitis A, hepatitis B, hepatitis C,herpes simplex type I, herpes simplex type II, human immunodeficiencyvirus, (HIV), human papilloma virus (HPV), influenza, measles, mumps,papova virus, polio, respiratory syncytial virus, rinderpest,rhinovirus, rotavirus, rubella, SARS virus, smallpox, viral meningitis,and the like. Infections diseases may also be caused by bacteriaincluding Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni,Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani,Diptheria, E. coli, Legionelia, Helicobacter pylori, Mycobacteriumrickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonas aeruginosa, S.pneumonia, Streptococcus, Staphylococcus, Vibria cholerae, Yersiniapestis, and the like. Infectious diseases may also be caused by fungisuch as Aspergillus fumigatus, Blastomyces dermatitidis, Candidaalbicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum, Penicillium marneffei, and the like. Infectious diseases mayalso be caused by protozoa and parasites such as chlamydia, kokzidioa,leishmania, malaria, rickettsia, trypanosoma, and the like.

Furthermore, Ep-CAM targeting proteins of the present invention may beused to prevent or treat additional conditions including but not limitedto heart conditions such as congestive heart failure (CHF), myocarditisand other conditions of the myocardium; skin conditions such as rosecea,acne, and eczema; bone and tooth conditions such as bone loss,osteoporosis, Paget's disease, Langerhans'cell histiocytosis,periodontal disease, disuse osteopenia, osteomalacia, monostotic fibrousdysplasia, polyostotic fibrous dysplasia, bone metastasis, bone painmanagement, humoral malignant hypercalcemia, periodontal reconstruction,spinal cord injury, and bone fractures; metabolic conditions such asGaucher's disease; endocrine conditions such as Cushing's syndrome; andneurological conditions.

Formulation

Pharmaceutical compositions are contemplated wherein an Ep-CAM targetingprotein of the present invention and and one or more therapeuticallyactive agents are formulated. Formulations of the Ep-CAM targetingproteins of the present invention are prepared for storage by mixing theEp-CAM targeting protein having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980,expressly incorporated by reference), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, acetate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyidiimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners andother flavoring agents; fillers such as microcrystalline cellulose,lactose corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium, metal complexes (erg.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG). In a preferred embodiment, thepharmaceutical composition that comprises the Ep-CAM targeting proteinof the present invention may be in a water-soluble form, such as beingpresent as pharmaceutically acceptable salts, which is meant to includeboth acid and base addition salts. “Pharmaceutically acceptable acidaddition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferred are the ammonium, potassium, sodium, calcium, and magnesiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. The formulations to be used for in vivo administration arepreferrably sterile. This is readily accomplished by filtration throughsterile filtration membranes or other methods.

The Ep-CAM targeting proteins disclosed herein may also be formulated asimmunoliposomes. A liposome is a small vesicle comprising various typesof lipids, phospholipids and/or surfactant that is useful for deliveryof a therapeutic agent to a mammal. Liposomes containing the Ep-CAMtargeting protein are prepared by methods known in the art, such asdescribed in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688;Hwang et at., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. Nos.4,485,045; 4,544,545; and PCT WO 97/38731, all expressly incorporated byreference. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556, expressly incorporated by reference. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. A chemotherapeutic agent or othertherapeutically active agent is optionally contained within the liposome(Gabizon et al., 1989, J National Cancer Inst 81:1484, expresslyincorporated by reference).

The Ep-CAM targeting protein and other therapeutically active agents mayalso be entrapped in microcapsules prepared by methods including but notlimited to coacervation techniques, interfacial polymerization (forexample using hydroxymethylcellulose or gelatin-microcapsules, orpoly-(methylmethacylate) microcapsules), colloidal drug delivery systems(for example, liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules), and macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol,A. Ed. 1980. Sustained-release preparations may be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymer, which matrices are in the form ofshaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot® (whichare injectable microspheres composed of lactic acid-glycolic acidcopolymer or lactic acid polymer and leuprolide acetate),poly-D-(−)-3-hydroxybutyric acid, and ProLease® (commercially availablefrom Alkermes, which is a microsphere-based delivery system composed ofthe desired bioactive molecule incorporated into a matrix ofpoly-DL-lactide-co-glycolide (PLG)).

Administration

Administration of the pharmaceutical composition comprising an Ep-CAMtargeting protein of the present invention, preferably in the form of asterile aqueous solution, may be done in a variety of ways, including,but not limited to orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, topically (eg., gels, salves, lotions,creams, etc.), intraperitoneally, intramuscularly, intrapulmonary,vaginally, parenterally, rectally, or intraocularly. In some instances,for example for the treatment of wounds, inflammation, etc., the EpCAMtargeting protein may be directly applied as a solution or spray. As isknown in the art, the pharmaceutical composition may be formulatedaccordingly depending upon the manner of introduction.

Subcutaneous administration may be preferable in some circumstancesbecause the patient may self-administer the pharmaceutical composition.Many protein therapeutics are not sufficiently potent to allow forformulation of a therapeutically effective dose in the maximumacceptable volume for subcutaneous administration. This problem may beaddressed in part by the use of protein formulations comprisingarginine-HCl, histidine, and polysorbate (see WO 04091658). Anti-Ep-CAMantibodies or Fc fusions of the present invention may be more amenableto subcutaneous administration due to, for example, increased potency,improved serum half-life, or enhanced solubility.

As is known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The Ep-CAM targeting proteins of the presentinvention may also be delivered using such methods. For example,administration may venious be by intravenous infusion with 0.9% sodiumchloride as an infusion vehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Ep-CAM targeting proteins of the presentinvention may be more amenable to intrapulmonary delivery. FcRn ispresent in the lung, and may promote transport from the lung to thebloodstream (e.g. Syntonix WO 04004798, Bitonti et.al. (2004) Proc. Nat.Acad. Sci. 101:9763-8, both expressly incorporated by reference).Accordingly, anti-Ep-CAM antibodes or Fc fusions that bind FcRn moreeffectively in the lung or that are released more efficiently in thebloodstream may have improved bioavailability following intrapulmonaryadministration. Ep-CAM targeting proteins of the present invention mayalso be more amenable to intrapulmonary administration due to, forexample, improved solubility or altered isoelectric point.

Furthermore, Ep-CAM targeting proteins of the present invention may bemore amenable to oral delivery due to, for example, improved stabilityat gastric pH and increased resistance to proteolysis. Furthermore, FcRnappears to be expressed in the intestinal epithelia of adults (Dickinsonet.al. (1999) J. Clin. Invest. 104:903-11), so anti-Ep-CAM antibodies orFc fusions of the present invention with improved FcRn interactionprofiles may show enhanced bioavailability following oraladministration. FcRn mediated transport of Ep-CAM targeting proteins mayalso occur at other mucus membranes such as those in thegastrointestinal, respiratory, and genital tracts (Yoshida et. al.(2004) Immunity 20:769-83).

In addition, any of a number of delivery systems are known in the artand may be used to administer the Ep-CAM targeting proteins of thepresent invention. Examples include, but are not limited to,encapsulation in liposomes, microparticles, microspheres (eg. PLA/PGAmicrospheres), and the like. Alternatively, an implant of a porous,non-porous, or gelatinous material, including membranes or fibers, maybe used. Sustained release systems may comprise a polymeric material ormatrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides,copolymers of L-glutamic acid and ethyl-L-gutamate, ethylene-vinylacetate, lactic acid-glycolic acid copolymers such as the Lupron Depot®,and poly-D-(−)-3-hydroxyburyric acid. It is also possible to administera nucleic acid encoding the Ep-CAM targeting protein of the currentinvention for example by retroviral infection, direct injection, orcoating with lipids, cell surface receptors, or other transfectionagents. In all cases, controlled release systems may be used to releasethe Ep-CAM targeting protein at or close to the desired location ofaction.

Dosing

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active Ep-CAM targeting proteinin the formulation may vary from about 0.1 to 100 weight %. In apreferred embodiment, the concentration of the Ep-CAM targeting proteinis in the range of 0.003 to 1.0 molar. In order to treat a patient, atherapeutically effective dose of the Ep-CAM targeting protein of thepresent invention may be administered. By “therapeutically effectivedose” herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.0001 to 100 mg/kg of bodyweight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight,with 1 to 10 mg/kg being preferred.

In some embodiments, only a single dose of the Ep-CAM targeting proteinis used. In other embodiments, multiple doses of the Ep-CAM targetingprotein are administered. The elapsed time between administrations maybe less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours,about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, ormore than 2 weeks.

In other embodiments the Ep-CAM targeting proteins of the presentinvention are administered in metronomic dosing regimes, either bycontinuous infusion or frequent administration without extended restperiods. Such metronomic administration may involve dosing at constantintervals without rest periods. Typically such regimens encompasschronic low-dose or continuous infusion for an extended period of time,for example 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or more.The use of lower doses may minimize side effects and the need for restperiods.

In certain embodiments the Ep-CAM targeting protein of the presentinvention and one or more other prophylactic or therapeutic agents arecyclically administered to the patient. Cycling therapy involvesadministration of a first agent at one time, a second agent at a secondtime optionally additional agents at additional times, optionally a restperiod, and then repeating this sequence of administration one or moretimes. The number of cycles is typically from 2-10. Cycling therapy mayreduce the development of resistance to one or more agents, may minimizeside effects, or may improve treatment efficacy.

Combination therapies

The Ep-CAM targeting proteins of the present invention may beadministered concomitantly with one or more other therapeutic regimensor agents. The additional therapeutic regimes or agents may be used toimprove the efficacy or safety of the Ep-CAM targeting protein. Also,the additional therapeutic regimes or agents may be used to treat thesame disease or a comorbidity rather than to alter the action of theEp-CAM targeting protein. For example, an Ep-CAM targeting protein ofthe present invention may be administered to the patient along withchemotherapy, radiation therapy, or both chemotherapy and radiationtherapy. The Ep-CAM targeting protein of the present invention may beadministered in combination with one or more other prophylactic ortherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, cytokines, growth inhibitory agents,anti-hormonal agents, kinase inhibitors, anti-angiogenic agents,cardioprotectants, immunostimulatory agents, immunosuppressive agents,agents that promote proliferation of hematological cells, angiogenesisinhibitors, protein tyrosine kinase (PTK) inhibitors, additional Ep-CAMtargeting proteins, FcγRIIb or other Fc receptor inhibitors, or othertherapeutic agents.

The terms “in combination with” and “co-administration” are not limitedto the administration of the prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the Ep-CAM targetingprotein of the present invention and the other agent or agents areadministered in a sequence and within a time interval such that they mayact together to provide a benefit that is increased versus treatmentwith only either the Ep-CAM targeting protein of the present inventionor the other agent or agents. It is preferred that the Ep-CAM targetingprotein and the other agent or agents act additively, and especiallypreferred that they act synergistically. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended. The skilled medical practitioner can determine empirically, orby considering the pharmacokinetics and modes of action of the agents,the appropriate dose or doses of each therapeutic agent, as well as theappropriate timings and methods of administration.

In one embodiment, the Ep-CAM targeting proteins of the presentinvention are administered with one or more additional moleculescomprising antibodies or Fc. The Ep-CAM targeting proteins of thepresent invention may be co-administered with one or more otherantibodies that have efficacy in treating the same disease or anadditional comorbidity; for example two antibodies may be administeredthat recognize two antigens that are overexpressed in a given type ofcancer, or two antigens that mediate pathogenesis of an autoimmune orinfectious disease.

Examples of anti-cancer antibodies that may be co-administered include,but are not limited to, anti 17-IA cell surface antigen antibodies suchas Panorex™ (edrecolomab); anti-4-1BB antibodies; anti-4Dc antibodies;anti-A33 antibodies such as A33 and CDP-833; anti-α4β1 integrinantibodies such as natalizumab; anti-α4β7 integrin antibodies such asLDP-02; anti-αVβ1 integrin antibodies such as F-200, M-200, and SJ-749;anti-αVβ3 integrin antibodies such as aboiximab, CNTO-95, Mab-17E6, andVitaxin™; anti-complement factor 5 (C5) antibodies such as 5G1.1;anti-CA125 antibodies such as OvaRex® (oregovomab); anti-CD3 antibodiessuch as Nuvion® (visilizumab) and Rexomab; anti-CD4 antibodies such asIDEC-151, MDX-CD4, OKT4A; anti-CD6 antibodies such as Oncolysin B andOncolysin CD6; anti-CD7 antibodies such as HB2; anti-CD19 antibodiessuch as B43, MT-103, and Oncolysin B; anti-CD20 antibodies such as 2H7,2H7.v16, 2H7.v114, 2H7.v115, Bexxar® (tositumomab), Rituxan®(rituximab), and Zevalin® (Ibritumomab tiuxetan); anti-CD22 antibodiessuch as Lymphocide™ (epratuzumab); anti-CD23 antibodies such asIDEC-152; anti-CD25 antibodies such as basiliximab and Zenapax®(daclizumab); anti-CD30 antibodies such as AC10, MDX-060, and SGN-30;anti-CD33 antibodies such as Mylotarg® (gemtuzumab ozogamicin),Oncolysin M, and Smart M195; anti-CD38 antibodies; anti-CD40 antibodiessuch as SGN-40 and toralizumab; anti-CD40L antibodies such as 5c8,Antova™, and IDEC-131; anti-CD44 antibodies such as bivatuzumab;anti-CD46 antibodies; anti-CD52 antibodies such as Campath®(alemtuzumab); anti-CD55 antibodies such as SC-1; anti-CD56 antibodiessuch as huN901-DM1; anti-CD64 antibodies such as MDX-33; anti-CD66eantibodies such as XR-303; anti-CD74 antibodies such as IMMU-110;anti-CD80 antibodies such as galiximab and IDEC-1 14; anti-CD89antibodies such as MDX-214; anti-CD123 antibodies; anti-CD138 antibodiessuch as 8-B4-DM1; anti-CD146 antibodies such as AA-98; anti-CD148antibodies; anti-CEA antibodies such as cT84.66, labetuzumab, andPentacea™; anti-CTLA-4 antibodies such as MDX-101; anti-CXCR4antibodies; anti-Ep-CAM antibodies such as ABX-EGF, Erbitux®(cetuximab), IMC-C225, and Merck Mab 425; anti-Ep-CAM antibodies such asCrucell's anti-Ep-CAM, ING-1, and IS-IL-2; anti-ephrin B2/EphB4antibodies; anti-Her2 antibodies such as Herceptin®, MDX-210; anti-FAP(fibroblast activation protein) antibodies such as sibrotuzumab;anti-ferritin antibodies such as NXT-211; anti-FGF-1 antibodies;anti-FGF-3 antibodies, anti-GFO8 antibodies; anti-FGFR antibodies,anti-fibrin antibodies; anti-G250 antibodies such as WX-G250 andRencarex®; antiG D2 ganglioside antibodies such as EMD-273063 andTriGem; anti-GD3 ganglioside antibodies such as BEC2, KW-2871, andmitumomab; anti-gpIIb/IIIa antibodies such as ReoPro: anti-heparinaseantibodies; anti-Her2/ErbB2 antibodies such as Herceptin® (trastuzumab),MDX-210, and pertuzumab; anti-HLA antibodies such as Oncolym®, Smart1D10; anti-HM1.24 antibodies: anti-ICAM antibodies such as ICM3;anti-IgA receptor antibodies; anti-IGF-1 antibodies such as CP-751871and EM-164; anti-IGF-1R antibodies such as IMC-A12; anti-IL-6 antibodiessuch as CNTO-328 and elsilimomab; anti-IL-15 antibodies such asHuMax™-IL15; anti-KDR antibodies; anti-laminin 5 antibodies; anti-LewisY antigen antibodies such as Hu3S193 and IGN-311; anti-MCAM antibodies;anti-Muc1 antibodies such as BravaRex and TriAb; anti-NCAM antibodiessuch as ERIC-1 and ICRT; anti-PEM antigen antibodies such as Theragynand Therex; anti-PSA antibodies; anti-PSCA antibodies such as IG8;anti-Ptk antbodies; anti-PTN antibodies; anti-RANKL antibodies such asAMG-162; anti-RLIP76 antibodies; anti-SK-1 antigen antibodies such asMonopharm C; anti-STEAP antibodies; anti-TAG72 antibodies such asCC49-SCA and MDX-220; anti-TGF-β antibodies such as CAT-152; anti-TNF-αantibodies such as CDP571, CDP870, D2E7, Humira® (adalimumab), andRemicade® (infliximab); anti-TRAIL-R1 and TRAIL-R2 antibodies;anti-VE-cadherin-2 antibodies; and anti-VLA-4 antibodies such asAntegren™. Furthermore, anti-idiotype antibodies including but notlimited to the GD3 epitope antibody BEC2 and the gp72 epitope antibody105AD7, may be used. In addition, bispecific antibodies including butnot limited to the anti-CD3/CD20 antibody Bi20 may be used.

Examples of antibodies that may be co-administered to treat autoimmuneor inflammatory disease, transplant rejection, GVHD, and the likeinclude, but are not limited to, anti-α4β7 integrin antibodies such asLDP-02, anti-beta2 integrin antibodies such as LDP-01, anti-complement(C5) antibodies such as 5G1.1, anti-CD2 antibodies such as BTI-322,MEDI-507, anti-CD3 antibodies such as OKT3, SMART anti-CD3, anti-CD4antibodies such as IDEC-151, MDX-CD4, OKT4A, anti-CD 11a antibodies,anti-CD14 antibodies such as IC14, anti-CD18 antibodies, anti-CD23antibodies such as IDEC 152, anti-CD25 antibodies such as Zenapax,anti-CD40L antibodies such as 5c8, Antova, IDEC-131, anti-CD64antibodies such as MDX-33, anti-CD80 antibodies such as IDEC-114,anti-CD147 antibodies such as ABX-CBL, anti-E-selectin antibodies suchas CDP850, anti-gpIIb/IIIa antibodies such as ReoPro/Abcixima,anti-ICAM-3 antibodies such as ICM3, anti-ICE antibodies such as VX-740,anti-FcR1 antibodies such as MDX-33, anti-IgE antibodies such asrhuMab-E25, anti-IL-4 antibodies such as SB-240683, anti-IL-5 antibodiessuch as SB-240563, SCH55700, anti-IL-8 antibodies such as ABX-IL8,anti-interferon gamma antibodies, and anti-TNFa antibodies such asCDP571, CDP870, D2E7, Infliximab, MAK-195F, anti-VLA4 antibodies such asAntegren. Examples of other Fc-containing molecules that may beco-administered to treat autoimmune or inflammatory disease, transplantrejection, GVHD, and the like include, but are not limited to, the p75TNF receptor/Fc fusion Enbrel® (etanercept) and Regeneron's IL-1 trap.

Examples of antibodies that may be co-administered to treat infectiousdiseases include, but are not limited to, anti-anthrax antibodies suchas ABthrax, anti-CMV antibodies such as CytoGam and sevirumab,anti-cryptosporidium antibodies such as CryptoGAM, Sporidin-G,anti-helicobacter antibodies such as Pyloran, anti-hepatitis Bantibodies such as HepeX-B, Nabi-HB, anti-HIV antibodies such asHRG-214, anti-RSV antibodies such as felvizumab, HNK-20, palivizumab,RespiGam, and anti-staphylococcus antibodies such as Aurexis, Aurograb,BSYX-A110, and SE-Mab.

Alternatively, the Ep-CAM targeting proteins of the present inventionmay be co-administered with one or more other molecules that compete forbinding to one or more Fc receptors. For example, co-administeringinhibitors of the inhibitory receptor FcγRIIb may result in increasedeffector function. Similarly, co-administering inhibitors of theactivating receptors such as FcγRIIIa may minimize unwanted effectorfunction. Fc receptor inhibitors include, but are not limited to, Fcmolecules that are engineered to act as competitive inhibitors forbinding to FcγRIIb FcγRIIIa, or other Fc receptors, as well as otherimmunoglobulins and specificially the treatment called IVIg (intravenousimmunoglobulin). In one embodiment, the inhibitor is administered andallowed to act before the Ep-CAM targeting protein is administered. Analternative way of achieving the effect of sequential dosing would be toprovide an immediate release dosage form of the Fc receptor inhibitorand then a sustained release formulation of the Ep-CAM targeting proteinof the invention. The immediate release and controlled releaseformulations could be administered separately or be combined into oneunit dosage form. Administration of an FcγRIIb inhibitor may also beused to limit unwanted immune responses, for example anti-Factor VIIIantibody response following Factor VIII administration to hemophiliacs.

In one embodiment, the Ep-CAM targeting proteins of the presentinvention are administered with a chemotherapeutic agent. By“chemotherapeutic agent” as used herein is meant a chemical compounduseful in the treatment of cancer. Examples of chemotherapeutic agentsinclude but are not limited to alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN™), alkyl sulfonates such as busulfan,improsulfan and piposulfan; androgens such as calusterone,dromostanolone propionate, epitiostanol, mepitiostane, testolactone;anti-adrenals such as aminoglutethimide, mitotane, trilostane;anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti estrogens includingfor example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, andtoremifene (Fareston); anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; folic acidreplenisher such as frolinic acid; nitrogen mustards such aschlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin. phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; proteins such asarginine deiminase and asparaginase; purine analogs such as fludarabine,6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and docetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (suchas Tomudex), additional chemotherapeutics including aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;difluoromethylornithine (DMFO); elformithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mritoxantrone, mopidamol; nitracrine; pentostatin;phenamet; pirarubicin; podophyllinic acid; 2ethylhydrazide;procarbazine; PSK®; razoxane; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa;chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;etoposide (VP-168); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11;retinoic acid; esperamicins; capecitabine.Pharmaceutically acceptable salts, acids or derivatives of any of theabove may also be used.

A chemotherapeutic or other cytotoxic agent may be administered as aprodrug, By “prodrug” as used herein is meant a precursor or derivativeform of a pharmaceutically active substance that is less cytotoxic totumor cells compared to the parent drug and is capable of beingenzymatically activated or converted into the more active parent form.See, for example Wilman, 1986, Biochemical Society Transactions, 615thMeeting Belfast, 14:375-382; and Stella et al., “Prodrugs: A ChemicalApproach to Targeted Drug Delivery, ” Directed Drug Delivery, Borchardtet al. (ed.): 247-267, Humana Press, 1985; both expressly incorporatedby reference. The prodrugs that may find use with the present inventioninclude but are not limited to phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, beta-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use with the Ep-CAM targetingproteins of the present invention include but are not limited to any ofthe aforementioned chemotherapeutic agents.

A variety of other therapeutic agents may find use for administrationwith the Ep-CAM targeting proteins of the present invention. In oneembodiment, the Ep-CAM targeting protein is administered with ananti-angiogenic agent. By “anti-angiogenic agent” as used herein ismeant a compound that blocks, or interferes to some degree, thedevelopment of blood vessels The anti-angiogenic factor may, forinstance, be a small molecule or a protein, for example an antibody, Fcfusion, or cytokine, which binds to a growth factor or growth factorreceptor involved in promoting angiogenesis. The preferredanti-angiogenic factor herein is an antibody that binds to VascularEndothelial Growth Factor (VEGF). Other agents that inhibit signalingthrough VEGF may also be used, for example RNA-based therapeutics thatreduce levels of VEGF or VEGF-R expression, VEGF-toxin fusions,Regeneron's VEGF-trap, and antibodies that bind VEGF-R. In an alternateembodiment, the Ep-CAM targeting protein is administered with atherapeutic agent that induces or enhances adaptive immune response, forexample an antibody that targets CTLA-4. Additional anti-angiogenesisagents include, but are not limited to, angiostatin (plasminogenfragment), antithrombin III, angiozyme, ABT-627, Bay 12-9566, benefin,bevacizumab, bisphosphonates, BMS-275291, cartilage-derived inhibitor(CDI), CAI, CD59 complement fragment, CEP-7055, Col 3, combretastatinA-4, endostatin (collagen XVIII fragment), farnesyl transferaseinhibitors, fibronectin fragment gro-beta, halofuginone, heparinases,heparin hexasaccharide fragment, HMV833, human chorionic gonadotropin(hCG), IM-862, interferon alpha, interferon beta, interferon gamma,interferon inducible protein 10 (IP-10), interleukin-12, kringle 5(plasminogen fragment), marimastat. metalloproteinase inhibitors (egTIMPs), 2-methodyestradiol, MMI 270 (CGS 27023A), plasminogen activiatorinhibitor (PAI), platelet factor-4 (PF4), prinomastat, prolactin 16kDafragment, proliferin-related protein (PRP), PTK 787/ZK 222594,retinoids, solimastat, squalamine, SS3304, SU5416, SU6668, SU11248,tetrahydrocortisol-S, tetrathiomolybdate, thalidomide, thrombospondin-1(TSP-1), TNP-470, transforming growth factor beta (TGF-β),vasculostatin, vasostatin (calreticulin fragment), ZS6126, and ZD6474.

In a preferred embodiment, the Ep-CAM targeting protein is administeredwith a tyrosine kinase inhibitor. By “tyrosine kinase inhibitor” as usedherein is meant a molecule that inhibits to some extent tyrosine kinaseactivity of a tyrosine kinase. Examples of such inhibitors include butare not limited to quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines;pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706;pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo(2,3-d) pyrimidines;curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide);tyrphostines containing nitrothiophene moieties; PD-0183805(Warner-Lambert); antisense molecules (e.g. those that bind toErbB-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396);tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787(Novartis/Schering A G); pan-ErbB inhibitors such as C1-1033 (Pfizer);Affinitac (ISIS 3521; Isis/Lilly); Imatinib mesylate (STI571, Gleevec®;Novartis); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); C1-1033(Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen); ZD6474 (AstraZeneca);PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); or as described inany of the following patent publications: U.S. Pat. No. 5,804,396; PCTWO 99/09016 (American Cyanimid); PCT WO 98/43960 (American Cyanamid);PCT WO 97/38983 (Warner-Lambert); PCT WO 99/06378 (Warner-Lambert); PCTWO 99/06396 (Warner-Lambert); PCT WO 96/30347 (Pfizer, Inc); PCT WO96/33978 (AstraZeneca); PCT WO96/3397 (AstraZeneca); PCT WO 96/33980(AstraZeneca), gefitinib (IRESSA™, ZD1839, AstraZeneca), and OSI-774(Tarceva™, OSI Pharmaceuticals/Genentech); all expressly incorporated byreference.

In another embodiment, the EP-CAM targeting protein is administered withone or more immunomodulatory agents. Such agents may increase ordecrease production of one or more cytokines, up- or down-regulateself-antigen presentation, mask MHC antigens, or promote theproliferation, differentiation, migration, or activation state of one ormore types of immune cells. Immunomodulatory agents include but notlimited to: non-steroidal anti-inflammatory drugs (NSAIDs) such asasprin, ibuprofed, celecoxib, diclofenac, etodolac, fenoprofen,indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin,rofecoxib, naproxen, ketoprofen, and nabumetone; steroids (eg.glucocorticoids, dexamethasone, cortisone, hydroxycortisone,methylprednisolone, prednisone, prednisolone, trimcinolone,azulfidineicosanoids such as prostaglandins, thromboxanes, andleukotrienes; as well as topical steroids such as anthralin,calcipotriene, clobetasol, and tazarotene); cytokines such as TGFb,IFNa, IFNb, IFNg, IL-2, IL-4, IL-10; cytokine, chemokine, or receptorantagonists including antibodies, soluble receptors, and receptor-Fcfusions against BAFF, B7, CCR2, CCRS, CD2, CD3, CD4, CD6, CD7, CD8,CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45,CD52, CD64, CD80, CD86, CD147, CD152, complement factors (C5, D) CTLA4,eotaxin, Fas, ICAM, ICOS, IFNα, IFNβ, IFNγ, IFNAR, IgE, IL-1, IL-2,IL-2R, IL-4, IL-5R, IL-6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15,IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGFβ, TNFα,TNFβ, TNF-R1, T-cell receptor, including Enbrel® (etanercept), Humira®(adalimumab), and Remicade® (infliximab); heterologous anti-lymphocyteglobulin; other immunomodulatory molecules such as 2-amino-6-aryl-5substituted pyrimidines, anti-idiotypic antibodies for MHC bindingpeptides and MHC fragments, azathioprine, brequinar, bromocryptine,cyclophosphamide, cyclosporine A, D-penicillamine, deoxyspergualin,FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide,malononitriloamides (eg. leflunomide), methotrexate, minocycline,mizoribine, mycophenolate mofetil, rapamycin, and sulfasasazine.

In an alternate embodiment, Ep-CAM targeting protein of the presentinvention are administered with a cytokine. By “cytokine” as used hereinis meant a generic term for proteins released by one cell populationthat act on another cell as intercellular mediators. Examples of suchcytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta,insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1IL-1alpha, IL-2, IL-3, IL-4IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture, and biologically active equivalents of the native sequencecytokines,

In a preferred embodiment, cytokines or other agents that stimulatecells of the immune system are co-administered with the Ep-CAM targetingprotein of the present invention. Such a mode of treatment may enhancedesired effector function. For example, agents that stimulate NK cells,including but not limited to IL-2 may be co-administered, In anotherembodiment, agents that stimulate macrophages, including but not limitedto C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine(Beigier-Bompadre et. al. (2003) Scand. J. Immunol, 57: 221-8, expresslyincorporated by reference), may be co-administered. Also, agents thatstimulate neutrophils, including but not limited to G-CSF, GM-CSF, andthe like may be administered. Furthermore, agents that promote migrationof such immunostimulatory cytokines may be used. Also additional agentsincluding but not limited to interferon gamma, IL-3 and IL-7 may promoteone or more effector functions.

In an alternate embodiment, cytokines or other agents that inhibiteffector cell function are co-administered with the Ep-CAM targetingprotein of the present invention. Such a mode of treatment may limitunwanted effector function,

In an additional embodiment, the Ep-CAM targeting protein isadministered with one or more antibiotics, including but not limited to.aminoglycoside antibiotics (eg. apramycin, arbekacin, bambermycins,butirosin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin,paromomycin, ribostamycin, sisomycin, spectrinomycin), aminocyclitols(eg. sprctinomycin), amphenicol antibiotics (eg. azidamfenicol,chloramphenicol, florfrnicol, and thiamphemicol), ansamycin antibiotics(eg. rifamide and rifampin), carbapenems (eg. imipenem, meropenem,panipenem); cephalosporins (eg. cefaclor, cefadroxii, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide,cefpirome, cefprozil, cefuroxine, cefixime, cephalexin, cephradine),cephamycins (cefbuperazone, cefoxitin, cefminox, cefmetazole, andcefotetan); lincosamides (eg. clindamycin, lincomycin); macrolide (eg,azithromycin, brefeldin A, clarithromycin, erythromycin, roxithromycin,tobramycin), monobactams (eg. aztreonam, carumonam, and tigernonam);mupirocin; oxacephems (eg. flomoxef, latamoxef, and moxalactam);penicillins (eg. amdinocillin, amdinocillin pivoxil, amoxicillin,bacampicillin, bexzylpenicillinic acid, benzylpenicillin sodium,epicillin, fenbenicillin, floxacillin, penamecillin, penethamatehydriodide, penicillin o-benethamine, penicillin O, penicillin V,penicillin V benzoate, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium); potypeptides (eg. bacitracin, colistin,polymixin B, teicoplanin, vancomycin); quinolones (amifloxacin,cinoxacin, ciprofloxacin, enoxacin, enrofloxacin, feroxacin, flumequine,gatifloxacin, gemifloxacin, grepafloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pefloxacin,pipemidic acid, rosoxacin, rufloxacin, sparfioxacin, temafloxacin,tosufloxacin, trovafloxacin); rifampin; streptogramins (eg.quinupristin, dalfopristin); sulfonamides (sulfanilamide,sulfamethoxazole); tetracyclenes (chlortetracycline, demeclocyclinehydrochloride, demethylchlortetracycline, doxycycline, duramycin,minocycline, neomycin, oxytetracycline, streptomycin, tetracycline,vancomycin).

Anti-fungal agents such as amphotericin B, ciclopirox, clotrimazole,econazole, fluconazole, flucytosine, itraconazole, ketoconazole,niconazole, nystatin, terbinafine, terconazole, and tioconazole may alsobe used.

Antiviral agents including protease inhibitors, reverse transcriptaseinhibitors, and others, including type I interferons, viral fusioninhibitors, and neuramidase inhibitors, may also be used. Examples ofantiviral agents include, but are not limited to, acyclovir, adefovir,amantadine, amprenavir, clevadine, enfuvirtide, entecavir, foscarnet,gangcyclovir, idoxuridine, indinavir, lopinavir, pleconaril, ribavirin,rimantadine, ritonavir, saquinavir, trifluridine, vidarabine, andzidovudine, may be used.

The Ep-CAM targeting proteins of the present invention may be combinedwith other therapeutic regimens. For example, in one embodiment, thepatient to be treated with an anti-Ep-CAM antibody or Fc fusion of thepresent invention may also receive radiation therapy. Radiation therapycan be administered according to protocols commonly employed in the artand known to the skilled artisan. Such therapy includes but is notlimited to cesium, iridium, iodine, or cobalt radiation. The radiationtherapy may be whole body irradiation, or may be directed locally to aspecific site or tissue in or on the body, such as the lung, bladder, orprostate. Typically, radiation therapy is administered in pulses over aperiod of time from about 1 to 2 weeks. The radiation therapy may,however, be administered over longer periods of time. For instance,radiation therapy may be administered to patients having head and neckcancer for about 6 to about 7 weeks. Optionally, the radiation therapymay be administered as a single dose or as multiple, sequential doses.The skilled medical practitioner can determine empirically theappropriate dose or doses of radiation therapy useful herein. Inaccordance with another embodiment of the invention, the Ep-CAMtargeting protein of the present invention and one or more otheranti-cancer therapies are employed to treat cancer cells ex vivo. It iscontemplated that such ex vivo treatment may be useful in bone marrowtransplantation and particularly, autologous bone marrowtransplantation. For instance, treatment of cells or tissue(s)containing cancer cells with Ep-CAM targeting protein and one or moreother anti-cancer therapies, such as described above, can be employed todeplete or substantially deplete the cancer cells prior totransplantation in a recipient patient.

Radiation therapy may also comprise treatment with an isotopicallylabeled molecule, such as an antibody. Examples ofradioimmunotherapeutics include but Zevalin™ (Y-90 labeled anti-CD20),LymphoCide™ (Y-90 labeled anti-CD22) and Bexxar™ (1-131 labeledanti-CD20)

It is of course contemplated that the Ep-CAM targeting proteins of theinvention may employ in combination with still other therapeutictechniques such as surgery or phototherapy.

A number of the receptors that may interact with the Ep-CAM targetingproteins of the present invention are polymorphic in the humanpopulation. For a given patient or population of patients the efficacyof the Ep-CAM targeting proteins of the present invention may beaffected by the presence or absence of specific polymorphisms inproteins. For example, FcγRIIIA is polymorphic at position 158, which iscommonly either V (high affinity) or F (low affinity). Patients with theV/V homozygous genotype are observed to have a better clinical responseto treatment with the anti-CD20 antibody Rituxan® (rituximab), likelybecause these patients mount a stronger NK response (Dall'Ozzo et. al.(2004) Cancer Res. 64:4664-9, expressly incorporated by reference).Additional polymorphisms include but are not limited to FcγRIIA R131 orH131, and such polymorphisms are known to either increase or decrease Fcbinding and subsequent biological activity, depending on thepolymorphism. Ep-CAM targeting proteins of the present invention maybind preferentially to a particular polymorphic form of a receptor, forexample FcγRIIIA 158 V, or to bind with equivalent affinity to all ofthe polymorphisms at a particular position in the receptor, for exampleboth the 158V and 158F polymorphisms of FcγRIIIA. In a preferredembodiment, Ep-CAM targeting proteins of the present invention may haveequivalent binding to polymorphisms may be used in an antibody toeliminate the differential efficacy seen in patients with differentpolymorphisms. Such a property may give greater consistency intherapeutic response and reduce non-responding patient populations. Suchvariant Fc with identical binding to receptor polymorphisms may haveincreased biological activity, such as ADCC, CDC or circulatinghalf-life, or alternatively decreased activity, via modulation of thebinding to the relevant Fc receptors. In a preferred embodiment, Ep-CAMtargeting proteins of the present invention may bind with higher orlower affinity to one of the polymorphisms of a receptor, eitheraccentuating the existing difference in binding or reversing thedifference. Such a property may allow creation of therapeuticsparticularly tailored for efficacy with a patient population possessingsuch polymorphism. For example, a patient population possessing apolymorphism with a higher affinity for an inhibitory receptor such asFcγRIIB could receive a drug containing an Ep-CAM targeting protein withreduced binding to such polymorphic form of the receptor, creating amore efficacious drug,

In a preferred embodiment, patients are screened for one or morepolymorphisms in order to predict the efficacy of the Ep-CAM targetingproteins of the present invention. This information may be used, forexample, to select patients to include or exclude from clinical trialsor, post-approval, to provide guidance to physicians and patientsregarding appropriate dosages and treatment options, For example, inpatients that are homozygous or heterozygous for FcγRIIIA 158F antibodydrugs such as the anti-CD20 mAb, Rituximab are minimially effective(Carton 2002 Blood 99: 754-758; Weng 2003 J. Clin. Oncol. 21:3940-3947);such patients may show a much better clinical response to the antibodiesof the present invention. In one embodiment, patients are selected forinclusion in clinical trials for an antibody of the present invention iftheir genotype indicates that they are likely to respond significantlybetter to an antibody of the present invention as compared to one ormore currently used antibody therapeutics. In another embodiment,appropriate dosages and treatment regimens are determined using suchgenotype information. In another embodiment, patients are selected forinclusion in a clinical trial or for receipt of therapy post-approvalbased on their polymorphism genotype, where such therapy contains anEp-CAM targeting protein engineered to be specifically efficacious forsuch population, or alternatively where such therapy contains an Ep-CAMtargeting protein that does not show differential activity to thedifferent forms of the polymorphism.

Included in the present invention are diagnostic tests to identifypatients who are likely to show a favorable clinical response to anEp-CAM targeting protein of the present invention, or who are likely toexhibit a significantly better response when treated with an Ep-CAMtargeting protein of the present invention versus one or more currentlyused antibody therapeutics. Any of a number of methods for determiningFcγR polymorphisms in humans known in the art may be used.

Furthermore, the present invention comprises prognostic tests performedon clinical samples such as blood and tissue samples. Such tests mayassay for effector function activity, including but not limited to ADCC,CDC, phagocytosis, and opsonization, or for killing, regardless ofmechanism, of cancerous or otherwise pathogenic cells. In a preferredembodiment, ADCC assays, such as those described previously, are used topredict, for a specific patient, the efficacy of a given Ep-CAMtargeting protein of the present invention. Such information may be usedto identify patients for inclusion or exclusion in clinical trials, orto inform decisions regarding appropriate dosages and treatmentregimens. Such information may also be used to select a drug thatcontains a particular Ep-CAM targeting protein that shows superioractivity in such assay.

EXAMPLES

Examples are provided below to illustrate the present invention, Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For reference toimmunoglobulin constant regions, positions are numbered according to theEU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda; expressly incorporated byreference), Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobutin families Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1

Anti-Ep-CAM Antibodies with Reduced Immunogenicity

FIGS. 1 and 2 provide some heavy and light chain variable regionsequences of the anti-Ep-CAM antibodies used in the present study. Themouse, parent chimeric heavy and light chains are labeled H0 17-1A andL0 17-1A respectively. Due to the wide use of hybridoma technology, asubstantial number of antibodies are derived from nonhuman sources.However, nonhuman proteins are often immunogenic when administered tohumans, thereby greatly reducing their therapeutic utility.Immunogenicity is the result of a complex series of responses to asubstance that is perceived as foreign, and may include production ofneutralizing and non-neutralizing antibodies, formation of immunecomplexes, complement activation, mast cell activation, inflammation,hypersensitivity responses, and anaphylaxis. Several factors cancontribute to protein immunogenicity, including but not limited toprotein sequence, route and frequency of administration, and patientpopulation. Immunogenicity may limit the efficacy and safety of aprotein therapeutic in multiple ways. Efficacy can be reduced directlyby the formation of neutralizing antibodies. Efficacy may also bereduced indirectly, as binding to either neutralizing ornon-neutralizing antibodies typically leads to rapid clearance fromserum. Severe side effects and even death may occur when an immunereaction is raised. Thus in a preferred embodiment, protein engineeringis used to reduce the immunogenicity of the Ep-CAM targeting proteins ofthe present invention.

In order to reduce the potential for immunogenicity of the anti-Ep-CAMproteins of the present invention, the immunogenicity of the anti-Ep-CAMantibody 17-1A was reduced using a method described in U.S. Ser. No.60/619,483, filed Oct. 14, 2004 and U.S. Ser. No. 11/004,590, entitled“Methods of Generating Variant Proteins with Increased Host StringContent and Compositions Thereof”, filed on Dec. 6, 2004. The methodsreduce the potential for immunogenicity by increasing the human stringcontent of the antibody through mutations. The heavy and light chainswith reduced potential for immunogenicity are named H1, H2, H3, H4, H5,H6, H2.1, H2.2, etc and L1, L2, L3, L4, L3.1, L3.2 etc and are shown inFIGS. 1 and 2. The heavy and light chains of the original antibody,17-1A, are referred to as H0 and L0.

Example 2

Combinations of the different heavy and light chains were expressed andthe resulting antibodies, with names such as H3L3, H3/L3 or H3_L3, werepurified and examined. Anti-Ep-CAM antibodies were expressed bytransient transfection of vectors encoding the heavy and light chainsinto 293T cells grown in 10% ultra low IgG fetal bovine serum with 1 rmMsodium pyruvate and 1× non-essential amino acids (Gibco®, InvitrogenHayward Calif.). Five days after transfection, the culture media wasremoved and ran through a protein A column (Pierce Biotechnology Inc,Rockford Md.). FIG. 4 shows typical yields of some Ep-CAM-bindingproteins. FIGS. 5 and 6 contain gels showing the heavy and light chainsof some purified antibodies of the present invention. The heavy chainsmay be made with any type of constant domain including, in humans, IgG1,IgG2 and hybrids comprising IgG1 and IgG2 as well as mouse constantdomains such as IgG1 and IgG2a, which may be referred to as mIgG1 andmIgG2a. The sequences of many of these heavy chains may be found in FIG.3. Data demonstrating the use of the hybrid IgG1, IgG2 heavy chain maybe found in FIGS. 4, 5 and 20.

Example 3

Binding Properties of Anti-Ep-CAM Humanized Antibodies

FIG. 7 shows a schematic representation of the AlphaScreen assay.Binding affinity of anti-Ep-CAM antibodies to the extracellular domainof Ep-CAM was measured using a quantitative and sensitive method,AlphaScreen™ assay. The AlphaScreen™ assay is a bead-basednon-radioactive luminescent proximity assay. Laser excitation of a donorbead excites oxygen, which if sufficiently close to the acceptor beadwill generate a cascade of chemiluminescent events, ultimately leadingto fluorescence emission at 520-620 nm. The AlphaScreen™ assay wasapplied as a competition assay for screening the antibodies. Wild-typeIgG1 Ep-CAM antibody was biotinylated and extracellular domain of Ep-CAMwas DIGylated by standard methods for attachment to streptavidin donorbeads and anti-DIG acceptor beads. In the absence of competinganti-Ep-CAM antibodies (unlabeled), wild-type antibody (biotinylated)and Ep-CAM (DIGylated) interact and produce a signal at 520-620 nm.Addition of untagged antibody competes with wild-type biotinylatedanti-Ep-CAM and DIGylated-Ep-CAM interaction reducing fluorescencequantitatively to enable determination of relative binding affinities.

FIGS. 8 to 3 show representative AlphaScreen™ data of various humanizedantibodies of the present invention. These figure show competitionAlphaScreen™ data in which tested antibody in this case, competes with areference antibody for the binding to Ep-CAM or an antibody bindingprotein. The binding of the humanized antibodies for the antigen,Ep-CAM, and protein A are shown on the left and right sides,respectively, of the FIG. 8. The results are also summarized in FIG. 9.Most humanized antibodies have an Ep-CAM binding affinity within 2-foldof the wild type. That is, they have between 0.5 and 2.0 fold increasesin binding affinity relative to the wild type. Fold increase valuesgreater than 1.0 demonstrate stronger binding than the wild type, 17-1A.FIGS. 10 to 13 show repeat measurements of the humanized antibodiesusing the AlphaScreen™ method.

FIGS. 14 to 16 show binding reactions for WT and variant anti-Ep-CAMantibodies measured with surface plasmon resonance. The kineticconstants for the binding of anti-Ep-CAM antibodies to Ep-CAM antigenwere determined using surface plasmon resonance-based measurements on aBIAcore 3000 instrument (Biacore, Uppsala, Sweden). For the data in FIG.14, the second and fourth CM5 sensor chip flow cells were coupled withrecombinant human Ep-CAM/Fc (R&D Systems, Minneapolis, Minn.) usingamine chemistry. Approximately 1000 and 6000 response units wererespectively immobilized. The first and third flow cells wereethanolamine blocked to serve as reference flow cells. Bindingexperiments were performed by injecting antibodies over the Ep-CAM/Fcand reference flow cells at varying concentrations ranging from 1 nM to1000 nM in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%Surfactant P20, filtered & degassed (HBS-EP, Biacore, Uppsala, Sweden)using KINJECT (2 minutes association, 2 minutes dissociation) at 50μl/minute. The sensor chip was regenerated with 10 mM glycine, pH 1.5.Data transformation was prepared by subtraction of blank injections(buffer without analyte) and y-transform prior to the injection start tozero using BlAevaluation software. The responses between 12.5 and 100 nMwere globally analyzed using a one to one interaction (Langmuir) bindingmodel. The corresponding association rate constant (k_(a)), dissociationrate constant (k_(d)), maximum analyte binding capacity (R_(max)), bulkrefractive index contribution (RI), equilibrium association constant(K_(A)), equilibrium dissociation constant (K_(D)), steady state bindinglevel (R_(eq)), and observed rate constant (k_(obs)) derived from thesefits are presented in FIG. 14, along with the chi-square (X²) valuewhich represents the closeness of fit between the fit curve and theactual data curve.

The data for the variants in FIG. 15 was collected similarly, but theresulting binding/dissociation curves were not fit to a particularbinding model. The binding strength is presented as the SPR signal, theresponse units, after a fixed time of flowing the analyte over thesurface. Stronger binding interactions yield a higher number of responseunits after this fixed time.

The SPR association/dissociation curves of three variants are shown inFIG. 16. Three different concentrations are shown for each variant andover-laid onto the data curves are fitted curve generated with a 1:1binding model containing a drifting baseline. The three curves for eachvariant were fit simultaneously to the model yielding one dissociationconstant describing the binding of the variant to Ep-CAM. For example,the variant H3.77/L3 showed a dissociation constant of 6.49e-8 M⁻¹ inthis assay.

Example 4

ADCC of Variants

Antibody-dependent cellular cytotoxicity measurements were done toassess the interaction of the antibodies of the present invention withcomponents of the immune system. First, the relative binding of ahumanized anti-Ep-CAM and trastuzumab to two different cell lines wasmeasured. FIG. 17 shows the binding of a humanized anti-Ep-CAM andtrastuzumab to the gastric carcinoma line, KATOIII, and the breastcancer line, SkBr3. Each cell line was dissociated using Accutase wash,resuspended and seeded at 50,000 cells per well of a 96-well plate.Cells were either treated with a secondary antibody-fluor (PE) conjugateor first treated with either trastuzumab or anti-Ep-CAM followed bysecondary mAb treatment. After 20 minutes of incubation on ice, therelative binding of each mAb was measured using a Guava Technologies™flow cytometry unit. The following histograms show the binding profileand mean fluorescence of each population consisting a total of 4000 cellcounts per histogram. The results show that about 3 times as much Ep-CAMis present on KATOIII cells than SkBr3 cells.

ADCC was measured using either the DELFIA® EuTDA-based cytotoxicityassay (Perkin Elmer) or LDH Cytotoxicity Detection Kit (RocheDiagnostic). Human PBMCs were purified from leukopacks using a ficollgradient. NK cells were isolated from human PBMCs using negativeselection and magnetic beads (Miltenyi Biotec). For europium-baseddetection, target cells were first loaded with BATDA at 1×10⁶ cells/mland washed 4 times. For both europium- and LDH-based detection, targetcells were seeded into 96-well plates at 10,000 cells/well, andopsonized using antibodies at the indicated final concentration. TritonX100 and PBMCs alone were typically run as positive and negativecontrols. Effector cells were added at 25:1 PBMCs:target cells or 4:1 NKCells:target cells, and the plate was incubated at 37° C. for 4 hrs.Cells were incubated with either Eu3+ solution or LDH reaction mixture,and fluorescence was measured using the Fusion Alpha-FP. Data werenormalized to maximal (triton) and minimal (PBMCs alone) lysis, and fitto a sigmoidal dose-response model.

FIGS. 18 to 24 display the results of ADCC assays of various anti-Ep-CAMantibodies. Improved, ie greater, levels of ADCC may be seen as either ashift in potency or efficacy. Improved potency of antibody is seen as aleft shift of an ADCC curve compared to a reference curve. The leftshift indicates that less antibody is required to achieve the samedegree of cytotoxicy as the reference antibody. In addition, improvedADCC may also be evident as improved efficacy, which is seen as anupward shift in the ADCC curve compared to a reference curve. The upwardshift indicates that the same amount of antibody produces a greaterdegree of cytotoxicity. Improvements in potency and efficacy may occursimultaneously or separately depending on the two antibodies beingcompared, the assay conditions (cell lines used, antibodyconcentrations, etc) and other factors.

For example, in FIG. 20 a the humanized anti-Ep-CAM antibody H3.77_L3 WTmay be used as a reference, wild-type, antibody. In comparison to thisreference H3.77_L3 S239D/I332E shows a large increase in potency in thatthe midpoint of the ADCC curve has shifted to lower antibodyconcentrations by about 0.5 log of antibody concentration (log antibodyconcentration =−1.0 vs −0.5 for the reference antibody). This antibodyalso shows an increase in efficacy, because at log antibodyconcentration of 1.0, it has about 30% cytoxicity whereas the referenceantibody has about 20% cytotoxicity. H3.77_L3 G236A shows increasedefficacy, but very little change in potency as it shows more cytoxicityat higher antibody concentrations, but very little change in themidpoint of its ADCC curve. Also indicated in FIG. 20 a is the improvedpotency of H3.77_L3 S239D/I332E compared to H3.77_L3 G236A with verylittle change in efficacy. Relative to the H3.77_L3 WT antibody, all ofthe other antibodies shown in FIG. 20 a have improved efficacy, improvedpotency, or an improvement in both efficacy and potency.

Example 5

Eq-CAM-binding Antibodies with Fc Substitutions

Ep-CAM-binding antibodies may comprise substitutions in the Fc region,or other regions, to optimize the antibody function. FIGS. 4, 5, 13, and20 to 23 comprise data of anti-Ep-CAM antibodies comprisingsubstitutions in the Fc domain. The substitutions S239D, I332E, G326A,L235G, G236R, A330Y, H268E affect binding to the Fcgamma receptors (SeeU.S. ser. No. 11/124620 entitled “Optimized Fc Variants”). Thesubstitutions P257L, P257N, V308F, V308Y, V279Y, and Q311 V affectbinding to FcRn (See PCT WO06053301A2 entitled “Fc Variants with alteredbinding to FCRN”). The altered Fc receptor binding and effector function(ADCC) of many variants are shown in FIGS. 13 and 20 to 23. Manyanti-Ep-CAM antibodies were found to have increased killing of LS180 andHT29 cells, particularly those that comprise the modifications S239D,I332E, G326A, L235G, G236R, A330Y, or H268E. The increased ADCC of thesevariants may be made in human or mouse Fc regions, including human IgG1,and hybrids of two different human IgG's, as shown for example in FIG.13.

Example 6

Effector Function—Glycoforms

The optimal anti-Ep-CAM clinical candidate may comprise an alteredglycoform. An Ep-CAM binding protein was expressed in Lec13 cells andpurified by the standard methods described herein, including protein Achromatography. This Lec 13 expressed antibody is a glycoform variant inthat it is defucosylated; it lacks the fucose residue on it N-linkedcarbohydrate moiety connected to Asn297. The purified protein is shownin FIG. 6 and shows the expected molecular weights of the heavy andlight chains. This defucosylated anti-Ep-CAM has stronger binding to theFc receptor, FcgammaRIIIa (Val variant), as shown in FIG. 25, lowerpanel. The defucosylated variant has an affinity of Kd=2.8*10⁻⁸ comparedto Kd=2.8*10-7 for the typically glycosylated form. The Kd measurementwere made with Surface Plasmon Resonance fixing the antibody on thesurface and flowing FcgammaRIIIA over the chip.

Example 7

Because of the sequence differences between the various human Fcreceptors, modifications to the Fc domain of antibodies can specificallymodulate their affinity for different human FcR's. The importance of thedifferent FcR's on different effector functions has been seen throughthe use of FcR knockout mice (Nimmerman and Ravetch 2005 Science310:1510-1512). The FcR affinity differences may potentially impactactivation of various immune effector cells, because different effectorcells have differential expression of each receptor (Pricop et al 2001 JImmunology 166:531-537, Samuelsson et al 2001 Science 19(291):484486.For example, neutrophil activation is influenced by the FcγRIIa/FcγRIIbratio (Van Mirre et al. 2006 Blood 108(2):584-590).

Additionally, immune complexes and FcγRIIa binding stimulate dendriticcell maturation, but FcγRIIb activity is know to suppress theirmaturation (Boruchov et al. Journal of Clinical Investigation115(10):2914-2923. Nimmerman and Ravetch, 2006 Immunity 24:19-28).

Additionally, phagocytosis by monocytes and macrophages may be initiatedby antibody binding to FcγRIIa (Hunter et al. Blood 91(5):1762-1768,Tridandapani et al 2002 Journal of Biological Chemistry277(7):5082-5089). FcγRIIb, on the other hand, may also bind theantibodies but FcγRIIb does not induce phagocytosis by monocytes andmacrophages. Therefore, FcγRIIb may passively inhibit phagocytosis bybinding antibody that otherwise may be available to bind to FcγRIIA aswell under go its more active inhibitory functions.

Additionally, FcγRIIIa is the important Fc receptor for causingactivation of NK cells. Alternatively, FcγRIIa and FcγRIIb are expressedon monocytes, macrophages, neutrophils, and dendritic cells, and some ofthese cell types are also known to express FcγRIIIa. It is well known inthe art that activation of these cell types can depend on the relativeexpression and/or activation of FcγRIIa compared to FcγRIIb, and thatcoactivation of FcγRIIb with FcγRIIa can decrease the activation viaFcγRIIa. We therefore determined the affinity of several Fc modifiedEp-CAM-targeting antibodies to several human FcR's.

To determine the affinity of various Fc modified Ep-CAM-targetingantibodies for human FcR, surface plasmon resonance experiments wereperformed on a Biacore 3000 instrument. Antibody was immobilized on aprotein A/G surface and purified forms of three human Fc receptors(FcγRI, FcγRIIIa, FcγRIIa and FcγRIIb) were added in the solution phaseas analyte. Global curve-fitting of a set of sensorgrams derived from aFcR concentration series was used to determine dissociation constants(Kd) between each variant and each of the FcRs included in the study.Note that the allotype of FcRIIIa used in the experiments was the 158Vform, and that of FcRIIa was the 131R form.

The Kd values for a series of Fc modified Ep-CAM-targeting antibodiesare shown in FIGS. 26 and 27. The affinity values show several importanttrends. First, variants containing the substitutions S239D, I332E andH268E all have increased affinity for FcγRIIIa relative to the wt IgG1control. These substitutions, individually or in combinations, have“Fold KD” (FIG. 27) values greater than one. For example, H3.77_L3 S239DIgG1 has a FcγRIIIa Fold KD value of 5.6, demonstrating that it has5.6-fold stronger binding to FcγRIIIa than the wild type. Antibodieswith these modifications also have increase affinity for the Fcreceptors FcγRIIa and FcγRIIb.

Of additional interest are variants containing the G236A substitutions.All of these variants have specifically enhanced affinity for FcγRIIa.G236A results in a specific enhancement of FcγRIIa binding compared toScyRIIb binding. Indeed, the RIIa/RIIb affinity ratio ofG236A-containing variants is systematically improved, having a−log(RIIa/RIIB) value of about 1.0. This value means that the variantshave about a full log, or 10-fold, increased binding for FcγRIIacompared to FcγRIIb. These variants will find utility in treatment ofEp-CAM expressing cancers, where monocytes, macrophages, neutrophils,and dendritic cells are important effector cells.

The effect of particular substitutions on specific FcR's is seen inEp-CAM-targeting proteins comprising different Fc domains. For example,FIGS. 26 and 27 show data collected with antibodies comprising eitherthe human IgG1 or a hybrid Fc comprising both IgG1 and IgG2 sequences.FIG. 3 shows the sequences of some Fc domains used herein.

Example 8

In alternate embodiments, other IgG allotypes may be used as Fc domainsin an Ep-CAM-targeting protein. Gm polymorphism is determined by theIGH1, IGH2, and IGH3 genes, which have alleles encoding allotypicantigenic determinants referred to as G1m, G2m, and G3m allotypes formarkers of the human IgG1, IgG2 and IgG3 molecules. FIG. 28 a providessome common allotypes, as is well known in the art. One or more of theseallotypic mutations could be made in either the IgG1 or hybridEp-CAM-targeting antibodies by incorporating substitution, asillustrated in FIG. 28 b.

1. A variant anti-Ep-CAM antibody comprising a variant human Fc domain,said variant human Fc domain comprising at least one modification thatalters binding of said antibody to an Fc receptor compared to a parenthuman Fc domain.
 2. The antibody of claim 1, wherein said at least onemodification alters binding to an Fcgamma receptor.
 3. The antibody ofclaim 2, wherein said at least one modification comprises at least onesubstitution selected from the group consisting of: 236A, 239D, 268E,298A, 298D, 326D, 326E, 330L, 330Y, 332E, 333A, 334A, and 396L, whereinthe numbering is according to the EU index in Kabat et al.
 4. Theantibody of claim 1, wherein said at least one modification includes analtered glycoform.
 5. The antibody of claim 4, wherein said at least onemodification includes defucosylation.
 6. The antibody of claim 1,wherein said at least one modification alters binding to the Fcreceptor, FcRn.
 7. The antibody of claim 6, wherein said modification isa substitution selected from the group consisting of: 250Q, 257L, 257N,311A, 311V, 428L, 434A, and 434Y, wherein the numbering is according tothe EU index in Kabat et al.
 8. A variant anti-Ep-CAM antibodycomprising at least one modification that alters an effector function ofsaid variant antibody compared to an unmodified anti-Ep-CAM antibody. 9.The antibody of claim 8, wherein said effector function of saidanti-Ep-CAM antibody is antibody-dependent cellular cytotoxicity (ADCC).10. The antibody of claim 9, wherein said anti-Ep-CAM antibody comprisesan Fc domain comprising at least one substitution selected from thegroup consisting of: 236A, 239D, 268E, 298A, 298D, 326D, 326E, 330L,330Y, 332E, 333A, 334A, and 396L, wherein the numbering is according tothe EU index in Kabat et al.
 11. The antibody of claim 8, wherein saidanti-Ep-CAM antibody comprises an altered glycoform.
 12. The antibody ofclaim 11, wherein said anti-Ep-CAM antibody comprises an Fc regionlacking a fucose moiety.
 13. The antibody of claim 8, wherein saideffector function is complement-dependent cytoxicity.
 14. The antibodyof claim 13, wherein said anti-Ep-CAM antibody comprises an Fc domaincomprising at least one substitution selected from the group consistingof: K326W, K326Y, and E333S, wherein the numbering is according to theEU index in Kabat et al.
 15. The antibody of claim 1, wherein saidmodification increases the affinity of said antibody for FcγRIIIacompared to a parent antibody.
 16. The antibody of claim 15, whereinsaid modification increases the affinity of said antibody for FcγRIIIaat least 2-fold compared to a parent antibody.
 17. The antibody of claim15, wherein said modification increases the affinity of said antibodyfor FcγRIIIa at least 5-fold compared to a parent antibody.
 18. Theantibody of claim 1, wherein said modification comprises a substitutionselected from the group consisting of: 239D and 332E, wherein thenumbering is that of the EU index in Kabat et al.
 19. The antibody ofclaim 1, wherein said modification decreases the affinity of saidantibody for FcγRIIIa compared to a parent antibody.
 20. The antibody ofclaim 19, wherein said modification decreases the affinity of saidantibody for FcγRIIIa by at least 10fold compared to a parent antibody.21. The antibody of claim 19, wherein said modification comprises asubstitution selected from the group consisting of: 235G and 236R,wherein the numbering is that of the EU index in Kabat et al.
 22. Theantibody of claim 1, wherein said modification increases theFcγRIIa:FcγRIIb specificity for said antibody.
 23. The antibody of claim22 wherein said modification increases the FcγRIIa:FcγRIIb specificityfor said antibody by at least
 2. 24. The antibody of claim 22, whereinsaid modification increases the FcγRIIa:FcγRIIb specificity for saidantibody by at least
 8. 25. The antibody of claim 22, wherein saidmodification increases the FcγRIIa:FcγRIIb specificity between 7 to 11.26. The antibody of claim 1, wherein said modification specificallyincreases maturation or activation of monocytes, macrophages,neutrophils, or dendritic cells by said antibody compared to activationof natural killer (NK) cells by said antibody.
 27. The antibody of claim26, wherein said modification specifically increases activation ofneutrophils by said antibody compared to activation of natural killer(NK) cells by said antibody.
 28. The antibody of claim 26, wherein saidmodification does not substantially increase activation of naturalkiller cells.
 29. The antibody of claim 26, wherein said modificationspecifically increases activation by said antibody of dendritic cells.30. The antibody of claim 1, wherein said modification increases bindingto an activating Fc receptor and does not increase binding to FcγRIIb.31. The antibody of claim 1, wherein said modification specificallyincreases monocyte or macrophage phagocytosis.